2,

Mechanistic and Physical Studies of Methane Monooxygenase from Methylococcuscapsulatus (Bath)

by

Katherine E. Liu

B.A., Cornell University (1989)

Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

at the

Massachusetts Institute of Technology

February, 1995

© 1995 Massachusetts Institute of Technology

All rights reserved

Signature of Author Department of Chemistry November 14, 1994

Certified by ------V U I/ Professor Stephen J. Lippard Thesis Supervisor /q~

Accepted by Professor Dietmar Seyferth Chairman, Departmental Committee on Graduate Students e-;.{ence . . :

-. .; 1QO D 2

This doctoral thesis has been examined by a Committee of the Department of Chemistry as follows:

Professor Alan Davison, Committee Chair

/'-- l' 6/O ProfessorStephen J. Lippard Arthur Amos Noyes Professor of Chemistry

PA fessor JoAnne Stubbe John G. Sheehan Professor of Chemistry

Z n P A n Professor 4wliam H. me Johnson 3 Acknowledgments

There are many people who helped make it possible for me to write this thesis. First and foremost, I must thank Steve Lippard for providing me the opportunity to join his research group at MIT. He allowed me to work independently, yet provided me much support and encouragement over the last five years. Studying the MMO system has led us down a variety of paths, and he was never hesitant to let me pursue new directions. I would also like to thank JoAnne Stubbe, William Orme-Johnson, and Alan Davison for serving on my thesis committee. I was fortunate to collaborate with may people away from MIT on several projects. Vickie DeRose and Brian Hoffman are responsible for many exciting EPR discoveries. Marty Newcomb fought along side us against the mechanistic dogma we encountered. My trips to the National Tritium Labeling Facility were made more enjoyable by Barrie Wilkinson, from Heinz Floss' group, Phil Williams, and Hiromi Morimoto. Ah, the excitement of what sandwich to order for lunch! Southern hospitality was provided by Vincent Huynh, Dale Edmondson, and Danli Wang at Emory University in Atlanta, GA, who introduced me to rapid freeze-quench techniques. Someday we will have to go Tapas Bar Hopping! Back at MIT, I wish to thank Ann Valentine for enthusiastically accepting to continue the mechanistic work on MMO. I also appreciate her help ill purifying protein and carrying out some kinetic experiments at the end. I am sure she saved me many months in producing this thesis! Thanks goes to Amy Rosenzweig, who worked with me on those grueling protein purfications in the early days, and to Thanos Salifoglou for his great fermentation efforts. Axel Masschelein expedited the stopped-flow work by assembling our equipment and designing our system software. I am also grateful to the other members of the MMO subgroup who have given me support along the way: David Coufal, Sonja Komar-Panicucci, and Andrew Feig. The above mentioned people made both my professional and personal life at MIT more enjoyable. There are many more names to include who have given me fond memories to take away with me. Fishing trips and Thanksgiving dinners were made special by Mike Keck and Ted Carnahan. I must thank Pieter Pil and Steve Brown for toughening me after the move to the second floor, for 4 participating in numerous waterfights (let's get Pieter!), and for providing daily banter upstairs, along with Megan McA'Nulty and Joyce Whitehead. Many thanks go to JoAnne Yun and Maria Bautista, who fed me the Hlawaiian pizza that made it possible for me to finish this thesis, followed by the 52's that made it possible for me to celebrate finishing this thesis. I am also grateful to Susanna Herold for her boomerangs and Kevin Ward for his brownies. Other fun times were provided by Jonathan Wilker (see you at Boston '96!), Rajesh Manchanda, and Deborah Zamble (thanks for help with the move, too). David Goldberg has provided much support during the last five years, and I am thankful for his friendship. I wish to express my appreciation toward my family: my father, Sally, Kim and Dave, David and Cheryl, and Mike and Wendy, all of whom, I am sure, were convinced I dropped off the face of the planet while writing this thesis. Believe it or not, I will actually leave MIT!! Finally, I must thank the person who has had by far the deepest impact on both my personal and professional life, John Protasiewicz. I am positive that I could not have made it this far without him. I would express amazement at how one person could have provided such a constant stream of love and support, but I know it all comes so natural for such a warm and kind individual (I am afraid, though, that you will never fully understand how much I love you). Mechanistic and Physical Studies of Methane

Monooxygenase from Methylococcus capsulatus (Bath)

by

Katherine E. Liu

Submitted to the Department of Chemistry on November 14, 1994 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Chemistry

ABSTRACT

In Chapter 1, progress in understanding the sMMO systems of both M. capsulatus (Bath) and M. trichosporium OB3b are reviewed. Physical parameters of the hydroxylase component determined from a variety of spectroscopic techniques are discussed. Interactions of the hydroxylase with the protein B and reductase components are outlined. Included in this discussion is how the other proteins affect both the physical characteristics and the reactivity of the hydroxylase. Studies to determine how the enzyme reacts with dioxygen in the presence and absence of substrate are presented, and mechanistic proposals for the hydroxylation reaction are given. The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) are investigated through potentiometric titrations in Chapter 2. The potentials were determined by EPR spectroscopic quantitation of the mixed valent form of the hydroxylase as a function of added sodium dithionite in the presence of electron transfer mediators. Addition of substrate has little effect on the potentials, but protein B causes a dimunition of both potentials. The presence of both protein B and reductase inhibits reduction of the diiron center in the absence of substrate. When the substrate propylene is added to this system, reduction is greatly facilitated. These results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase. Chapter 3 describes studies the sMMO system by ENDOR spectroscopy. Information about the coordination sphere of the iron atoms both in the absence and presence of substrates and inhibitors is presented. Most significantly, these studies identify a proton resonance from the bridging hydroxide ligand present in the mixed-valent form of the hydroxylase. In Chapter 4, studies in which five mechanistic probes were used as substrates to investigate the possible formation of radical intermediates in the catalytic cycle of the sMMO system are outlined. No ring-opened products were observed, which suggests that the hydroxylation reaction may not proceed through substrate radical formation. A lower limit of 1013 s-1 was calculated for the rate constant of a radical rebound process to account for the 6 products recovered in the reaction. The possibility of a stereoelectronic barrier to ring opening in the active site of the enzyme is addressed through a semi-quantitative analysis of the rate constant with Marcus theory. Kinetic isotope effect experiments were also carried out. No intermolecular isotope effect was observed, which is consistent with C-H bond breaking not being involved in the rate determining step of the enzymatic reaction. Intramolecular kinetic isotope effects of kH/kD = 5 were obtained, indicating that the hydroxylation step of the reaction involves a substantial C-H bond stretching component. These results are discussed in terms of several possible detailed mechanisms for the MMO hydroxylase reaction. In Chapter 5, the use of chiral, tritiated hydrocarbons as substrates with MMO is described. These studies indicate that the stereoselectivity of the hydroxylation reaction depends on whether hydroxylation occurs at the proton or deuterium atom of the chiral carbon. A significant intermolecular kinetic isotope effect of kH/kD = 4 was obtained in most cases. The intramolecular kinetic isotope effects were much lower, and even inverted in some cases. An exchange mechanism in which the product alcohol reacts further in the active site prior to dissociation is invoked to reconcile the different stereoselectivity and the kinetic isotope effects observed with two enantiomers of the same substrate. Mechanistic implications for the overall hydroxylation reaction are discussed. The reactivity of the reduced hydroxylase protein (Hred) toward dioxygen is outlined in Chapter 6. Included in this work are the effects of protein B and the reductase on the regioselectivity, product yield, and rate constant of the hydroxylation reaction. Two intermediates in this reaction, designated L and Q, were observed. Kinetic parameters from optical stopped- flow and rapid freeze-quench M6ssbauer data indicate that Hred reacts with dioxygen initially to form L, which is subsequently converted to Q. The decay of Q occurs with concomitant production of the product alcohol and oxidized hydroxylase (Hox). The Mdssbauer parameters for both intermediates are unusual and do not match the values of any known diiron carboxylate- bridged model compounds. Based on an isomer shift of 0.66 mm/s, XmaxofO 625 nm, and an 1 8 -sensitive resonance Raman feature at 902 cm-1, L is assigned as a diiron(II) peroxide species. Intermediate Q exhibits a low isomer shift -=0.15 mm/s and is consistent with an Fe(IV) species. Proposed hydroxylation mechanisms are discussed in view of these results.

Thesis Supervisor: Dr. Stephen J. Lippard Title: Arthur Amos Noyes Professor of Chemistry 7

Table of Contents

Page Acknowledgments ...... 3 Abstract ...... 5 Table of Contents...... 7 List of Tables ...... 12 List of Figures ...... 14

Chapter 1. Studies of the Soluble Methane Monooxygenase Protein System: Structure,Component Interactions, and Hydroxylation Mechanism Introduction ...... 20 Structural Studies of sMMO Hydroxylase ...... 21 Component Interactions ...... 24 Mechanism of Hydroxylation ...... 30 Reaction of Hred with Dioxygen...... 30 Reaction of Q with Substrate...... 35 Conclusions ...... 40 References ...... 41 Tables...... 48 Figu res ...... 52

Chapter 2. Redox Properties of the Hydroxylase Component of Methane Monooxygenase from Methylococcus capsulatus (Bath) -- Effects of Protein B, Reductase, and Substrate Introduction...... 64 Experimental...... 66 Protein Isolation...... 66 Redox Titrations ...... 66 EPR Spectroscopy...... 67 Results ...... 68 Redox Titrations ...... 68 Component Interactions ...... 69 Discussion...... 70 Hydroxylase Potentials...... 70 Literature Comparisons ...... 72 8

Structural Implications ...... 72 Component Interactions...... 74 References...... 78 Tables ...... 81 Figu res ...... 83

Chapter 3. Electron Nuclear Double Resonance Studies of the Dinuclear Iron Center in the Hydroxylase Component of Methane Monooxygenase from Methylococcus capsulatus (Bath) Introduction ...... 98 Basics of EPR and ENDOR Spectroscopy ...... 99 Experimental...... 101 Native Samples...... 101 15 N Enrichment ...... 102 5 7Fe Enrichment ...... 102

D20 Exchange...... 103 17 H 2 0 Exchange ...... 103 Sample Preparation ...... 103 Addition of Protein B...... 104 Addition of Inhibitors/Substrates ...... 104 ENDOR Spectroscopy...... 105 Results and Discussion...... 105 EPR Spectrum ...... 105 1H ENDOR Spectra...... 105 14 N and 15 N ENDOR Spectra ...... 107 57Fe ENDOR Spectra ...... 109 Samples with Isotopically-Labeled DMSO ...... 109 Other Samples...... 110 Conclusions...... 111 R eferences ...... 112 Tables ...... 1...... 15 Figures ...... 116

Chapter 4. Radical Clock Substrate Probes and Kinetic Isotope Effect Studies of the Hydroxylation of Hydrocarbons by Methane Monooxygenase Introduction ...... 147 Experimental...... 149 9

Isolation and Purification of Proteins...... 149 Synthesis and Characterization of Substrates and Products ...... 150 Enzymatic Reactions...... 152 Instrumentation ...... 152 Product Identification and Quantitation ...... 153 Isotope Effect Studies...... 154 Results ...... 155 Reactions with 1...... 156 Reactions with 2...... 157 Reactions with 3...... 157 Reactions with 4...... 157 Reactions with 5...... 158 Discussion ...... 159 Analysis of Products Formed...... 159 Deuterium Isotope Effects...... 161 Mechanistic Considerations ...... 163 Formation of the Dioxygen-Activated Diiron Center ...... 163 The Hydroxylation Reaction...... 165 Comparisons with Related Work...... 172 Conclusions ...... 173 Acknowledgments...... 174 Referen ces...... 175 Tables ...... 182 Figures ...... 185

Chapter 5. Tritiated Chiral Alkanes as Substrates for Methane Monooxygenase from Methylococcus capsulatus (Bath): Probes for the Mechanism of Hydroxylation Prefa ce...... 211 Introduction...... 211 Experim enta l...... 214 Bacterial Growth and Fermentation ...... 214 Reagent Synthesis...... 216 (S)-[l- 1H,1-2H]-Ethanol ...... 216 (S)-[1-1H,1-2 H]-Ethyl Tosylate ...... 216 (S)-[1-2H,1- 3H]-Ethane ...... 217 Enzymatic Reactions...... 217 10

Derivitization...... 218 Product Analysis...... 219 Results ...... 219 Analysis of Lypholysates ...... 219 Analysis of Derivatives ...... 220 Discussion...... 223 Analysis of Product Distributions ...... 223 Alkanes are not Highly Restricted in the Active Site...... 223 H,T Vs D,T Products and Exchange at the -Carbon ...... 224 Kinetic Isotope Effects...... 227 Mechanistic Implications...... 229 Comparisons with Literature...... 231 Concluding Remarks...... 232 References ...... 234 Tables ...... 237 Figures ...... 243

Chapter 6. Intermediates in, and Kinetic Studies of the Reaction of Reduced Hydroxylase of MMO from Methylococcus capsulatus (Bath) with Dioxygen. Introduction ...... 290 Experimental ...... 292 Bacterial Growth and Protein Purification ...... 292 G eneration of H red...... 293 Single Turnover Reactions...... 294 Kinetic Studies...... 294 Product Distributions and Yields...... 294 KNl of Nitrobenzene ...... 297 Stopped-Flow Spectrophotometry ...... 297 Rapid Freeze-Quench Studies ...... 299 EPR Spectroscopy...... 299 M6ssbaiuer Spectroscopy...... 299 Resonance Raman Spectroscopy...... 300 Results...... 300 Studies of Nitrobenzene Hydroxylation ...... 300 Product Distributions and Yields in Single Turnover Expts ...... 300 Kinetic Analysis of the Hydroxylation of Nitrobenzene ...... 301 11

Intermediates Formed in the Reaction of Hred with Dioxygen...... 302 Spectroscopic Characterization of Intermediates ...... 302 Kinetic Analysis ...... 303 Discussion...... 305 MMO Component Interactions...... 305 Spectroscopic Studies of Intermediates ...... 310 Conclusions ...... 318 Acknowledgments...... 319 References...... 320 Tables ...... 324 F igu res ...... 328 Biography...... 381 12

List of Tables

Chapter 1. Table 1. Physical Properties of Hox from M. trichosporium OB3b and M. capsulatus(Bath) ...... 48 Table 2. Reduction Potentials of the MMO Hydroxylase from M. capsulatus (Bath) and M. trichosporium OB3b...... 49 Table 3. Product Distributions from Reactions of MM() with Nitrobenzene...... 50 'Table 4. Spectroscopic and Kinetic Parameters for Intermediates in the Reactions of Hred with Dioxygen ...... 51

Chapter 2. Table 1. Reduction Potentials of the MMO Hydroxylase from M. capsulatus (Bath) and M. trichosporiumOB3b Under Various Conditions ...... 81 Table 2. Reduction Potentials of the Mediators Employed in the Titrations ...... 82

Chapter 3. Table 1. 1H Hyperfine Coupling Constants for Hmx and Hmv with DMSO...... 115 Table 2. 14N and 15 N Coupling Constants of Hmv with and without DMSO ...... 115

Chapter 4. Table 1. Instrumental Conditions for GC and GC/M S Analyses ...... 182 Table 2. Retention Times of Reactants and Products in the Reaction of 1 - 5 with MMO Hydroxylase ...... 183 Table 3. Expected Products and Observed Ratios for Reactions with MMO from M. capsulatus(Bath) ...... 184

Chapter 5. Table 1. Component Amounts Used In Enzymatic Reactions at NTLF ...... 237 13

Table 2. Results of MMO Reactions with (S) and (R)-[1-2H ,1-3H ]-Ethane ...... 238 Table 3. Results of MMO Reactions with (S) and (R)-j[1-2H ,1- 3H ]-Butane ...... 239 Table 4. Results of MMO Reactions with (R), (S), and Racemic [2-3H]-Butane ...... 240 Table 5. Foul Sets of Conditions for which Quantitative Exchange Reactions are Calculated...... 241

Chapter 6. Table 1. Product Distributions from Reactions of MMO with Nitrobenzene ...... 324 Table 2. Mossbauer Parameters of Species Detected in Rapid Freeze Quench Samples from the Reaction of Hred with Dioxygen...... 325 Table 3. Rate Constants for Formation and Decay of Intermediate Q Under a Variety of Conditions...... 326 Table 4. Activation Parameters from Arrhenius and Eyring Plots of the Reaction of Hred with Dioxygen...... 327 14

List of Figures

Chapter 1. Figure 1. Active Site Structure of Hox as Determined by X-ray Crystallography ...... 52 Figure 2. Possible Structures for a Diiron(III) Peroxide Unit in Intermediate L...... 54 Figure 3. Proposed Route for Conversion of Intermediate L to Intermediate Q...... 56 Figure 4. Proposed Catalytic Cycle for the Hydroxylation of Hydrocarbons by MMO...... 58 Figure 5. Possible Mechanisms for Hydroxylation by sMMO...... 60

Chapter 2. Figure 1. X-band EPR Spectrum of Mixed-Valent Hydroxylase...... 83 Figure 2. X-band EPR Spectrum of Reduced Hydroxylase...... 85 Figure 3. EPR Spectra of the Hydroxylase at Different Reduction Potentials from a Reductive Titration...... 87 Figure 4. Plot Quantitating the Amount of Hydroxylase Present in Each Oxidation State with Conditions A...... 89 Figure 5. Plot Quantitating the Amount of Hydroxylase Present in Each Oxidation State with Conditions B and C...... 91 Figure 6. EPR Spectra of the Hydroxylase at Various Potentials in the Presence of Protein B and Reductase...... 93 Figure 7. X-band EPR Spectrum of Mixed-Valent Hydroxylase Formed Under Conditions B...... 95

Chapter 3. Figure 1. Energy Level Diagram of a Free Electron in an Applied Magnetic Field...... 116 Figure 2. Energy Level Diagram of a Paramagnetic Center Further Split by the Influence of a Nuclear Spin...... 118 Figure 3. Examples of ENDOR Spectra for Differing A Strengt hs...... 120 15

Figure 4 Q-band EPR Spectra of Hmv...... 122 Figure 5. Q-band EPR Spectra of Hmv in the Presence of DMSO...... 124 Figure 6. 1H ENDOR Spectra of Hmv at Various g-Values...... 126 Figure 7. 1H ENDOR Spectra of Hmv at g = 1.94...... 128 Figure 8. 1H ENDOR Spectrum of Hmv in the Presence of DMSO at g = 1.87...... 130 Figure 9. 14 N and 15N ENDOR Spectra of H m v...... 132 Figure 10. 1 4N and 15 N ENDOR Spectra of Hmv in the Presence of DMSO...... 134 Figure 11. 57 Fe ENDOR Spectra of the Fe(III) Site at Various g Values...... 136 Figure 12. 57Fe ENDOR Spectra of the Fe(II) Site at Various g Values ...... 138 Figure 13. ENDOR Spectra of Hmv in the Presence of Isotopically-Labeled DMSO...... 140 Figure 14. Mims Pulsed ENDOR Spectra of Hmv in the Presence of Isotopically-Labeled DMSO...... 142 Figure 15. 2H ESEEM Spectra of Hmv in the Presence of DMSO...... 144

Chapter 4. Figure 1. Reactions after Abstraction of a Hydrogen Atom from 1...... 185 Figure 2. Substrates 1 through 5...... 187 Figure 3. Typical GC Traces from Reactions of MMO with Probes 1 and 2...... 189 Figure 4. Formation of Product Alcohol over Time...... 191 Figure 5. Formation of 2a over Time...... 193 Figure 6. Product Formation of Reactions of MMO w ith 4...... 195 16

Figure 7. Proposed Catalytic Cycle for the Hydroxylation of Hydrocarbons by MMO...... 197 Figure 8. Possible Reactions of a Diferric Peroxide Intermediate in the Catalytic Cycle...... 199 Figure 9. Possible Modes of Peroxide Binding ...... 201 Figure 10. Possible Mechanisms for the Hydroxylation Reaction...... 203 Figure 11. Reaction Pathways after Abstraction of a Hydrogen Atom from 5...... 206 Figure 12. Plot of kr as a Function of the Exergonicity of the Ring-Opening Reaction...... 208

Chapter 5. Figure 1. Possible Reaction of MMO with (S)-[1-2H,1-3H]-Ethane...... 243 Figure 2. Reaction Apparatus Used at NTLF...... 245 Figure 3. Possible Reaction Products from Reactions with MMO and (S)-[1-2H,1- 3H]-Ethane ...... 247 Figure 4. 1H NMR Spectrum of the Lypholysate from the Reaction of MMO with (S)-[1-2H,1- 3H]-Ethane ...... 249 Figure 5. 3H NMR Spectrum of the Lypholysate from the Reaction of MMO with (S)-[1-2H,1- 3H]-Ethane ...... 251 Figure 6. Reaction Scheme of the Derivitization Procedure...... 253 Figure 7. 3H NMR Spectrum of the Derivitized Lypholysate from the Reaction of MMO with (S)-[1-2H,1- 3H]-Ethane ...... 255 Figure 8. 3H NMR Spectrum of the Derivitized Lypholysate from the Reaction of MMO with (S)-[1-2 H,1-3H]-Ethane (Zoom) ...... 257 Figure 9. 3H NMR Spectrum of the Lypholysate from the Reaction of MMO with (S)-[1-2H,1-3H]-Butane ...... 259 Figure 10. 3H NMR Spectrum of the Derivitized Lypholysate from the Reaction of MMO with (S)-[1-2H,1- 3H]-Butane ...... 261 Figure 11. 3H NMR Spectrum of the Derivitized Lypholysate from the Reaction of MMO with (S)-[1-2H,1- 3H]-Butane (Zoom) ...... 263 Figure 12. 3H NMR Spectrum of the Derivitized Lypholysate from the Reaction of MMO with (S)-[2-3H]-Butane (Zoom) ...... 265 17

Figure 13. 3H NMR Spectrum of the Derivitized Lypholysate from the Reaction of MMO with Racemic [2-3 H]-Butane (Zoom) ...... 267 Figure 14. Possible Alcohol Products after Reactions of (R)-[2-3H]-Butane with MMO ...... 269 Figure 15. Hypothetical Orientation of (S)-[1-2H,1-3 H]-Ethane in the Active Site...... 271 Figure 16. Hypothetical Orientation of (S)-[1-2 H,1-3 H]-Butane in the Active Site...... 273 Figure 17. Hypothetical Orientation in the Active Site of a Product Alcohol which is Undergoing and Exchange Reaction...... 275 Figure 18. Exchange Reaction Interconverting the H,T Products ...... 277 Figure 19. Exchange Reaction Interconverting the D,T Products ...... 279 Figure 20. Illustration of the Dependence of C2/C1 on k H/k D ...... 281 Figure 21. Effects of the Exchange Reaction on the Intermolecular Kinetic Isotope Effect...... 283 Figure 22. Two Possible Mechanisms for Hydroxylation by MMO...... 285 Figure 23. Precursors to the Four Products Observed by 3H NMR Spectroscopy...... 287

Chapter 6. Figure 1. Schematic of the Rapid Freeze-Quench Apparatus ...... 328 Figure 2. HPLC Traces of Single Turnover Reactions of Hred with Nitrobenzene and Dioxygen...... 330 Figure 3. Plot of the Yield of Single Turnover Reactions as a Function of Protein B Concentration ...... 332 Figure 4. Kinetic Analysis of the Catalytic Reaction of the MMO System with Nitrobenzene ...... 334 Figure 5. Kinetic Traces at 404 nm of Single Turnover Reactions with and without Protein B...... 336 Figure 6. Plot of Rate Constants of Single Turnover Reactions as a Function of Protein B Concentration ...... 338 Figure 7. M6issbauer Analysis of Intermediate L...... 340 18

Figure 8. Mdssbauer Spectrum of a 3 s Rapid Freeze-Quench Sample...... 342 Figure 9. Mbssbauer Analysis of Intermediate Q...... 344 Figure 10. Mtssbauer Spectra of Hox and Hred ...... 346 Figure 11. High Field M6ssbauer Spectra of fIred, L, and Q...... 349 Figure 12. Kinetic Trace of Intermediate L at 625 nm ...... 351 Figure 13. Optical Spectrum of Q over Time ...... 353 Figure 14. EPR Spectrum of a 155 ms Rapid Freeze-Quench Sample...... 355 Figure 15. EPR Spectrum of a 8 s Rapid Freeze-Quench Sample...... 357 Figure 16. Resonance Raman Spectra of Rapid Freeze-Quench Samples from the Reaction of Hred with Dioxygen...... 359 Figure 17. Resonance Raman Spectra of L with Excitation at 647 nm...... 361 Figure 18. Plots of the Percent M6ssbauer Area of L, Q, and Hox as a Function of Time ...... 363 Figure 19. Protein B Dependence of the Kinetic Traces of the Reaction of Hred with Dioxygen at 420 nm ...... 365 'Figure 20. Plot of the Rate Constants for Q Formation and Decay Vs Dioxygen Concentration ...... 367 Figure 21. Plot of the Rate Constants for Q Formation and Decay Vs pH ...... 369 Figure 22. Kinetic Trace of the Reaction of D20-Exchanged Hred with Dioxygen at 420 nm...... 371 Figure 23. Arrhenius and Eyring Plots for the Rate Constants for the Formation and Decay of Q...... 373 Figure 24. Kinetic Trace of the Reaction of Hred with Dioxygen in the Presence of Methane at 420 nm ...... 375 Figure 25. Proposed Structures for Intermediates L and Q...... 377 Figure 26. Proposed Catalytic Cycle for the Oxidation of Hydrocarbons by MMO...... 379 19

CHAPTER 1.

Studies of the Soluble Methane Monooxygenase Protein

System: Structure, Component Interactions, and Hydroxylation Mechanism. 20 Introduction Methanotrophic bacteria have the fascinating ability to use methane as their sole source of carbon and energy.1 These organisms play a crucial role in the atmospheric balance of this greenhouse gas and in the overall global carbon cycle by annually consuming billions of tons of methane. 2'3 Because these bacteria are capable of converting methane into methanol at ambient pressure and temperature, methanotrophs have received much attention in the search for alternative methods for methanol synthesis,4 which are currently not cost efficient and require high temperatures and pressures. 5 In addition to methane, these organisms can metabolize a wide variety of other substrates,6 -8 which has spurred interest in their bioremedial applications. 91 3 Methanotrophs rely on the enzymatic system methane monooxygenase (MMO) for their first metabolic step, as shown in equation (1).1,14 The MMO systems in two organisms, a Type I methanotroph 15 Methylococcuscapsulatus (Bath) and a Type II methanotroph 15 Methylosinus trichosporium OB3b, have

+ CH4 + 0: + H+ + NADH - CH3OH + H20 + NAD (1) been the focus of intense research in recent years. Progress toward understanding the structure of the system, the interactions between the three components, and the hydroxylation mechanism are reviewed here. In most methanotrophs, two distinct forms of MMO can exist in the cell, with the dominant species depending on the bioavailability of copper.16 The particulate MMO (pMMO) is a membrane bound system which has been difficult to study because of its instability outside the cell. Recent progress has been made in the isolation and characterization of pMMO from M. capsulatus (Bath), the activity of which was found to depend on the copper concentration within the 21 membrane.171 8 EPR and magnetic susceptibility studies indicate that pMMO contains an exchange-coupled trinuclear Cu cluster.18 Since the EPR signal of the chemically reduced tricopper cluster changed to that of the oxidized signal upon exposure to dioxygen, this unit is believed to occur at the active site of pMMO.18 Under conditions of copper stress, some methanotrophs can express a cytosolic, soluble form of MMO (sMMO),19-22 the properties of which form the focus of this review. The sMMO systems from both M. capsulatus (Bath) and M. trichosporiumOB3b have been well studied, and comprise three separate protein components which have all been purified to homogeneity.23' 24 The hydroxylase component, a 251 kD protein, contains two copies each of three subunits in an ca2P2Y2configuration. The c subunit of the hydroxylase contains a dinuclear iron center25 responsible for dioxygen activation and for hydroxylation of the substrate. 26 The 38.6 kD reductase contains an FAD and an Fe2S2 cofactor27 which enable it to relay electrons from NADH to the diiron center in the hydroxylase.2 8 2 9 The third component, protein B, contains no cofactors.30 This small, 15.5 kD protein interacts with hydroxylase and reductase in several ways. Structural Studies of sMMO Hydroxylase The hydroxylase component of sMMO belongs to a family of proteins which contain a non-heme, carboxylate-bridged, dinuclear iron unit at their active site.31 -34 Included in this family are the proteins hemerythrin, ribonucleotide reductase, and purple acid phosphatase. Understanding how the diiron unit is tuned in each protein to exhibit such diverse functionality, ranging from the reversible binding of dioxygen in hemerythrin to activation of dioxygen for converting methane to methanol in MMO, is a primary goal of research in this area. Emerging features of the non-heme systems can be compared to related structural and functional information about the analogous heme proteins hemoglobin and cytochrome P-450, which have been more extensively studied. 22 The diiron center in the sMMO hydroxylase has been investigated by a variety of physical techniques. Properties of the hydroxylase from both M. capsulatus (Bath.)and M. trichosporiumOB3b in three oxidation states are listed in Table 1. The enzyme is isolated in its native Fe(III)Fe(III) oxidation state (Hox).35 M6ssbauer parameters are consistent with the presence of two high spin iron(III) atoms,36 37 and are slightly perturbed with increased pH.37 Because of antiferromagnetic coupling between the Fe(III) atoms, the ground state of Hox is EPR silent.3 6 3 7 EPR signals at g = 4.3 and 2.0, together which account for < 5 % of total iron, are present in spectra of the enzyme in all oxidation states and are assigned to adventitious Fe(III) and a protein-associated free radical, respectively. The S = 2 excited state of Hox displays an integer spin EPR signal at

-' 3 7 g = 8 (J = -8 cm , where H-= -2JS1S2). Since the oxo-bridged diiron(III) units in hemerythrin and the R2 subunit of ribonucleotide reductase exhibit coupling constants of -135 and -110 cm 1, respectively, the small J value suggested that an oxo-bridge is not present in Ho. 37 This hypothesis was in accord with previous EXAFSspectral studies36 38 in which no short Fe-O distance was found and with the lack of any optical features above 300 nm in any oxidation state.36 A 2.2 A resolution x-ray crystal structure of Hox from M. capsulatus (Bath)39 is consistent with the occurrence of a bridging hydroxide ligand (vide infra), and the active site structure derived from this analysis is illustrated in Figure 1. Two additional bridges, a bidentate glutamate, and an exogenous acetate from the crystallization conditions, were also identified. The Fe...Fe distance was determined to be 3.4 A. Each iron atom is ligated by one histidine nitrogen atom. The remaining coordination sites are occupied by two monodentate glutamate residues on one iron atom, whereas the second iron atom contains one monodentate glutamates and a water ligand in its coordination sphere. 23 The mixed-valent Fe(II)Fe(III) oxidation state (Hmv) is readily accessible by one-electron reduction of the dinuclear center. Mbssbauer data indicate the presence of one Fe(III) and one Fe(II) center.37 Hmv gives rise to a rhombic EPR signal with gav = 1.8326,35 (J = -30 cm-1),36'3 7 which is characteristic of the EPR signals from the other mixed-valent non-heme carboxylate bridged diiron proteins such as hemerythrin (J = -15 cm-1).31 ENDOR spectroscopic studies of Hm,, from M. capsulatus (Bath) provided direct evidence for a hydroxide bridge and a terminal water ligand of the diiron unit.4 0 The hyperfine parameter of the hydroxide proton was highly unusual, displaying an extremely large and anisotropic coupling with the iron center. Exchange with D20 demonstrated that the proton was derived from solvent. Spectra with semimetazido hemerythrin displayed a similar feature, and confirmed the assignment since it is known from a variety of other methods to contain a bridging hydroxide ligand.41 4 4 A pulsed EPR report of Hmv from M. trichosporium OB3b was consistent with these conclusions.45 Extensive ESEEM and ENDOR studies of Hmv have been carried out.46-48 These experiments provided extensive characterization of the diiron core examining 14 N, 15 N, 1H, 2 H, 13C, and 57 Fe nuclei in native Hmv. The inhibitor DMSO4 8 provided information about the geometry of the substrate 46 48 binding site in Hmv. ' Mims pulsed ENDOR of Hmv treated with d6-DMSO suggested that the methyl groups bind the diiron unit asymmetrically.46 Further reduction of Hmv results in formation of the fully reduced, Fe(II)Fe(II) protein (Hred). Mbssbauer features of Hred from both organisms are best fit as two unique quadrupole doublets.3 74 9 For M. trichosporiumOB3b, the doublets were interpreted as indicating the presence of two distinct Fe(II) sites in the diiron center.3 7 For M. capsulatus (Bath), one doublet was attributed to dinuclear Fe(II) centers in the protein that react with dioxygen in a non- productive manner.49 Hred from M. trichosporiumnOB3b and both forms of Hred 24 from M. capsulatus (Bath) exhibit an EPR feature at g = 1526,36arising from a ferromagnetically coupled, S = 4 spin system, with the two lowest spin levels being nearly degenerate.5 0 Identification of nitrogen donor ligands was demonstrated through pulsed ENDOR and ESEEM spectra of Hred from M. capsulatus (Bath).51 These studies provided the first advanced EPR study of a non-Kramers doublet spin system, and suggest the possibility of investigating similar signals from other proteins in this family. EXAFS spectroscopy did not exhibit Fe...Fe backscattering in Hred.36 This observation could result from thermal disorder, or it may suggest that, as a consequence of the reduction of Hox to Hred, the Fe...Fe distance has increased significantly. MCD experiments indicate that both iron atoms are five-coordinate and are ferromagnetically coupled with J = 0.3 to 0.5 cm-1.5 2 Comparison with deoxyhemerythrin, which is antiferromagnetically coupled (J -12 to -36 cm-1)41' 42 5 3 5 4 and contains a hydroxide bridge, led to the hypothesis that the hydroxide bridge present in Hox and Hmv is converted to a water bridge in Hred.52 Component Interactions Although the structure of the hydroxylase is now reasonably well understood, less is known about the interactions among the three component proteins of MIMO. Despite the fact that the physical properties of the M. capsulatus (Bath) and M. trichosporium OB3b hydroxylase are very similar, preliminary work with the other components indicates that significant differences exist. The interactions among the component proteins are quite complex, as manifest by the regulation of electron transfer to the hydroxylase, the product yields and regioselectivity of the hydroxylation reaction, and the detailed kinetic behavior of the systems. In the M. capsulatus (Bath) system, all three components are necessary to obtain turnover with NADH as the reductant.5 5 With the M. trichosporiumOB3b 25 system, protein B is apparently not necessary.26 Instead, in this latter system protein B increases the initial rates of the catalytic hydroxylation reaction.26 Catalysis can be achieved by means of a shunt pathway with hydrogen peroxide and Hox,,alone from both organisms.56-58 The efficiency of the shunt pathway varies significantly, however. With M. trichosporiumOB3b, alcohol yields greater than those obtained with the complete reconstituted system were observed.5 6 Upon addition of protein B, however, the initial rates were diminished, behavior opposite to that observed with the catalytic system.59 Reasons for the different effects of protein B on the two reactions with Hox are unclear. With Hox from M. capsulatus (Bath), activities of only = 10 % of the values observed under optimal catalytic conditions were observed with the H202 shunt pathway, assuming specific activities greater than 200 mU/mg. 5 7 Since such poor yields were observed, the effect of protein B on that system was not investigated. Investigations of the reduction potentials of the diiron center in the hydroxylase described by equation (2) as well as the effects of adding the other

E1 ° E2 Fe(III)Fe(III) - Fe(II)Fe(III) - Fe(II)Fe(II) (1) proteins on the potentials, again demonstrated inconsistent behavior for the two organisms, as shown in Table 2. For M. capsulatus (Bath), values of +350 and -25

° 0 mV vs NHE E1 and E2 obtained from redox titrations measuring appearance and disappearance of the EPR signal of Hmv were initially reported. 3 5 To investigate problems attaining high yields of Hmv, these potentials were remeasured. Two sets of electron transfer mediators were used, yielding potentials of +48 and -135 mV vs NHE for one set of conditions,6 0 and +100 and -100 mV using the second set of conditions.6 1 Substrate had little effect on the potentials, whereas addition of protein B altered them to values of +50 and -170 26 mV. The presence of both protein B and reductase prevented the detection of EPR signals associated with Hmv or Hred at potentials as negative as - 200 mV.6 0 When the titration of the three components was carried out in the presence of substrate, only Hred was detected, but at potentials as high as 100 mV. This result indicates that, with the complete reconstituted system including substrate, 0 two-electron transfer occurs. Reduction Hox,,directly to Hred indicates that E2 has shifted to a value greater than E1°. No EPR signal indicative of Hmv was observed implying that Hmv is not physiologically significant. The inhibition of hydroxylase reduction by protein B and reductase is consistent with kinetic work i.nwhich this component prevented NADH oxidation by the MMO system in the absence of substrate.62 Addition of substrate similarly led to efficient electron transfer to the hydroxylase as measured by the consumption of NADH. ° E1 and E2 values of +76 and +21 mV, respectively, were measured by similar methods for Hox from M. trichosporium OB3b.63 Addition of protein B lowered the potentials to -52 mV and -115 mV. The regulation of electron transfer to the hydroxylase with protein B and reductase observed with the M. capsulatus (Bath) MMO was not seen with this system. Instead, it was reported that the potentials of Hox and of Hox with added protein B were shifted slightly :more positive in the presence of reductase, and the reduction was not substrate- dependent. As indicated by the negative shifts in the reduction potentials of HoX,61'63 protein B can affect with the diiron center in Hmv from both MMO systems. Consistent with this interpretation are EPR studies of Hmv from both organisms which indicate that in the presence of protein B, the EPR signal moves from gav

1.83 to gayv 1.75.46,64 The distribution of product formed by hydroxylation of nitrobenzene and isopentane with MMO from M. trichosporiumOB3b were investigated,56 59 and 27 the results with nitrobenzene are listed in Table 3. Protein B had a dramatic effect on the regioselectivity of hydroxylation.59 This behavior was attributed to changes in the hydroxylase structure upon complexation with protein B, causing the substrate molecule to interact differently at the active site of the enzyme. Similar studies of the substrate nitrobenzene with the M. capsulatus(Bath) system were carried out,4 9 and the results of this work are included in Table 3. Very different regioselectivity was observed with the native reconstituted system, with product distributions that curiously matched those obtained by the hydrogen peroxide shunt pathway with M. trichosporiumOB3b. With the M. capsulatus (Bath) system the presence of protein B in single turnover reactions with chemically reduced Hred had no effect on the regioselectivity of the reaction. This result implies that the reductase has a significant effect on the distribution of products from the hydroxylation reaction. Of interest are the KM values for nitrobenzene in the various systems. KM value of = 5 mM were obtained with 4 9 both the catalytic system from M. capsulatus (Bath) and with the H20 2 shunt pathway with. M. trichosporiun OB3b.56 As indicated above, these two systems display similar product distributions in the hydroxylation reaction. The catalytic system from M. trichosporium OB3b yielded a KM value = 100 PM, 56 however, and the regioslectivity also differed from that observed with the other two systems. Yields of single turnover reactions with chemically reduced Hre,,dfrom M. trichosporium OB3b were not sensitive to the presence of the other two components. 26 From these and other studies with this system, it was concluded that protein B does not affect the reactivity of the hydroxylase with dioxygen, and that it has little effect on the hydroxylation reaction once Hred is formed.64 In contrast to this work, experiments with MMO from M. capsulatus (Bath) indicated that total yield from single turnover reactions of chemically reduced 28 Hred was significantly affected by the other components of the system (see Chapter 6).49 Maximal yields occurred when greater than 1.5 equivalents of protein B were added, either prior to or following reduction of Hox to Hred. Combining both protein B and reductase with Hred resulted in slightly diminished yields (see Chapter 6), and adding protein B and reductase to Hox prior to reduction resulted in extremely low levels of hydroxylation. This latter finding is consistent with the redox studies in which, even at quite negative potentials, Hred was not detected by EPR spectroscopy with protein B and reductase present.6 0 Adding reductase alone to Hox or Hred did not have a substantial effect on the product yields, which were quite low since protein B was not present. Protein B from M. capsulatus (Bath) affects the hydroxylation in two ways. Studies of single turnover reactions reveal that protein B increases the hydroxylation efficiency reflected in increased yields and rate constants. As determined by the redox studies, it regulates electron transfer so as to occur only in the presence of substrate, which prevents the wasteful cconsumption of reducing equivalents and protects the enzyme from inactivation. The redox results also indicate that incubation of Hox with protein B and reductase produces a more reactive Hred species. The reactive Hred species formed in this manner could presumably be produced by chemical reduction of Hox with all three protein components as well as substrate present. The presence of nitrobenzene, the substrate in these experiments, prevented the mediated reduction of Hox to Hred, however.60 Addition of protein B and reductase to Hred may not produce the same reactive species as formed when Hox is reduced in the presence of protein B and reductase. For example, structural alterations produced along with the change in oxidation state from Hox to Hred could arise 29 from kinetic effects imposed by the other two components which influence the coordination sphere of the iron atoms.49 Protein B from M. capsulatus(Bath) not only increased the product yields, but it also influenced the rate constant in single turnover reactions of Hred with nitrobenzene. 4965 The pseudo-first order rate constant increased by up to 33- fold when Hred was titrated with protein B. Neither addition of reductase to Hox or Hred, nor of protein B and reductase to Hred, could similarly affect the rate constant. These observations again imply either that protein B alone activates the hydroxylase or that protein B and reductase do not significantly affect Hred. Complex formation among the three protein components of the M. trichosporium OB3b MMO has been demonstrated. 6 4 In this work, it was hypothesized that protein B binds tightly to Hox to form an activated complex but that, after formation of Hred, the binding affinity is diminished.6 364 Results reported for M. capsulatus (Bath) are consistent with initial binding of Hox to protein B to form an activated complex,4 9 because increased yields and rate constants are seen when protein B is added to Hred. If the Hred-protein B complex of M. capsulatus (Bath) is the species influencing reactivity, then the addition of more protein B, which would ensure that Hred is in the protein B- bound complex, would be expected to increase the rate constants. Since H is a dimer (227Y2'),two equivalents of protein B and reductase are believed to bind to one H molecule. Concentrations of protein B above two equivalents per Hred may diminish the rate constant of the reaction by sterically hindering substrate binding to Hred. The total product yield could still remain high since nearly all of the Hred is in the active protein B-bound form. If there are a finite number of binding sites for additional molecules of protein B beyond two equivalents, excess protein B would have little effect once all of these extra sites were fully occupied, except to block possibly the reductase binding site. This reasoning was 30 also invoked in work with M. trichosporiumOB3b to explain the titration curve describing the effect of protein B on the initial rates of the catalytic system with Iox and reductase.64 The M. capsulatus (Bath) reductase alters the distribution of products formed in the reaction of Hred and dioxygen with nitrobenzene, 49 but protein B plays a wider variety of roles in this MMO system. As mentioned earlier, it regulates electron transfer from the reductase to the hydroxylase so as to occur only in the presence of substrate,60 62 it shifts the reduction potentials of the substrate/reductase/hydroxylase complex,60 and it affects kobs and yields in single turnover reactions of Hred.4 9 Protein B from M. trichosporium OB3b changes product distributions with Hred,59 increases the initial velocity of the 64 complete reconstituted system, but reduces the initial velocity with the H202 shunt system.64 Reductase played no major role in the regioselectivity of the hydroxylation reaction in this case.59 With hydroxylase from both organisms, protein B perturbs the EPR signal of Hmv46'64 and lowers the reduction potentials.61 63 A unifying explanation for these observed functions has yet to be determined. Some of the differences may arise from variations in the proteins themselves. For example, appreciable differences in protein B from the two MMO's have been reported by the inability of antibodies raised toward one protein B to bind the other.66 More structural work on component interactions is essential to understand fully the fundamental reasons for the observed effects and to reconcile differences between the two MMO systems. Mechanism of Hydroxylation Reaction of Hred with Dioxygen. Hred is the form of the hydroxylase which reacts with dioxygen. 26 Recently, intermediates were detected in this reaction.67-70In all cases, dioxygen was mixed with Hred in the presence of two equivalents of protein B under 31 pseudo-first order conditions in dioxygen. Reactions were carried out at 4 °C in the absence of substrate to accumulate detectable quantities of the intermediates. Spectroscopic parameters and kinetic constants for two intermediates, designated L and Q, are listed in Table 4. The proposed reaction summary is given in equation (3). Magnetic Mdssbauer4 9'6 7 and EPR spectroscopy4 9 68 indicated that

k, k2 k3 Hrc.d L > Q ox (3)

both intermediates are diamagnetic. In the M. trichosporiumOB3b MMO system, rapid freeze-quench EPR studies revealed that the g = 15 EPR signal of Hred decays with a first order rate constant of = 22 + 5 sO- upon exposure to dioxygen.68 Based on this observation, a proposed intermediate named "P" was suggested as a precursor to Q (vide invfa). M6ssbauer analysis of rapid freeze- quench samples prepared from the M. capsulatus(Bath) system identified such an intermediate,6 9 but this compound was named "L" since it was unknown at that time whether it corresponed to the immediate precursor to Q. Kinetic Mossbauer data for L from M. capsulatus (Bath) indicates that L forms with a first order rate constant of = 25 s-1,68and concomitantly with the decay of Hred. The minimal mechanism therefore dictates that intermediate L represents the initial species formed in the reaction of Hred with dioxygen. The M6ssbauer spectrum of L consists of a symmetric doublet which was fit with parameters = 0.66 + 0.02 mm/s and AEQ = 1.51 + 0.03 mm/s. 6 9 The isomer shift is larger than values (0.45 - 0.55 mm/s) generally seen for carboxylate-bridged diiron(III) clusters but significantly smaller than isomer shift values for diiron(II) clusters (1.1 - 1.3 mm/s).3 1 -33 Although parameters matching the values of L have not been observed in model compounds, the 32 magnitude of the isomer shift is close to parameters seen for diiron(III) clusters, and L was accordingly proposed to be a diiron(III) peroxide complex.69 Such species have been assigned to products formed in reactions of several diiron(II) model complexes with dioxygen and result from a two electron transfer from the Fe atoms to the dioxygen ligand.34 The optical absorption at 625 nm ( = 500 M- 1 cm-1) reported for L70,71 is close to reported electronic spectral maxima (max 600 or 604 nm) for several diiron(III) peroxide model complexes.7 2 7- 4 Similarly, the resonance Raman spectrum of L with excitation at 647 nm displayed a band at 902 cm-1,70 which matches u(O-O) values reported for several model compounds. 27- 7 4 From the sharp, symmetrical shape of the Mbssbauer signal, the iron atoms were interpreted as being in nearly equivalent coordination environments.6 9 To account for the high isomer shift value of 0.66 mm/s for L, six-coordinate iron atoms with considerable peroxide-to-iron charge transfer or seven coordinate iron atoms were invoked.69 These structures could arise from the two pentacoordinate iron atoms in Hred transferring two electrons to oxygen to form a peroxide ligand. The two resulting Fe(II) atoms may bind the peroxide ligand either a t-r12, 11,1 1 or an 12,112 fashion. This latter binding mode has been observed for oxyhemocyanin7 5 and in a dicopper(II) peroxide complex.7 6 Figure 2 illustrates possible structures for L which are compatible with its spectroscopic parameters.49 The time dependence revealed by rapid freeze-quench M6ssbauer experiments with M. capsulatus (Bath)4 9 indicated that decay of L proceeds with the concomitant formation of another intermediate, named compound "Q". This intermediate, observed both in the M. trichosporium OB3b67'68 and the M. capsulatus (Bath)4 9'6 9 MMO systems by M6ssbauer and optical spectroscopy, decays faster in the presence of substrates. This behavior indicates that this 33 intermediate is on the kinetic reaction pathway for hydroxylation.49,68 The M6ssbauer spectrum of Q from M. trichosporiumOB3b contained a doublet with 6 = 0.17 mm/s and AEQ = 0.53 mm/s.67 This doublet was symmetric and the iron atoms were assumed reside in equivalent coordination environments. In the M. capsulatus (Bath) system, two unresolved doublets of equal intensity were distinguished and fit to the following parameters: 6 = 0.21 + 0.02 mm/s and AEQ

= 0.68 ± 0.03 mm/s for doublet 1, and 6 = 0.14 + 0.02 mm/s and AEQ = 0.55 ± 0.03 mm/s for doublet 2.69 The average isomer shift and quadrupole splitting of the two doublets, 0.18 mm/s and 0.62 mm/s, respectively, agrees with parameters obtained from the corresponding spectrum of Q from the M. trichosporiumOB3b organism. In the M. capsulatus (Bath) system, however, the presence of two distinct signals for Q indicates that the iron atoms are in inequivalent sites. From the average isomer shift value of 0.18 mm/s, it was suggested that Q is an iron(IV) oxo species.6 7 Figure 3 illustrates possible structures for Q. In M. capsulatus (Bath), the inequivalent Fe sites could arise from rearrangement of the protein ligands in Q compared to their positions as revealed in the x-ray crystal structure of Hox.39 Alternatively, the oxo ligand in the proposed Fe(IV) species may not be bound symmetrically between the Fe atoms. Equivalent iron sites in the M. trichosporiumOB3b system could arise from symmetrical bridging of the oxygen atoms between the two Fe(IV) atoms.4 9

Cleavage of the 0-0 bond in L followed by release of H20 most likely results in the formation of Q.49 The second iron atom in the MMO hydroxylase active site can stabilize this unit through redox charge delocalization in a similar fashion to the stabilization of a high valent iron oxo species in cytochrome P-450 by oxidation of the porphyrin ring to a 7r-cation radical.7 7 A large change in

1 -1 entropy (ASt -: 147 Jmole- K ) was reported for the conversion of L to Q, consistent with the release of a water molecule during this step.49 The rate 34 constant for the growth of Q was independent of dioxygen concentration.49' 68 Since formation of intermediate L is rapid and irreversible and formation of Q is the slow step, the rate constant for Q is not expected to be dependent on dioxygen concentration under experimentally practical conditions. Conversion of intermediate L, a diiron(III) peroxide species, to Q, a putative diiron(IV) oxo species, implies proton transfer to the core such that a molecule of water is produced from the second oxygen atom of the peroxide. The lack of a pH dependence or a significant kinetic deuterium isotope effect on the rate constant for this process indicates that proton transfer is not overall rate determining in the conversion.4 9 The widely accepted hydroxylation mechanism for the heme protein cytochrome P-450 invokes an Fe(IV) oxo species which abstracts a hydrogen atom from the substrate molecule.77 Since cytochrome P-450 cannot hydroxylate methane, a similar Fe(IV) oxo intermediate in MMO could be significantly more reactive. Very recently, Fe(IV) intermediates have been ruled out in the mechanism for generating the tyrosyl radical in the R2 subunit of ribonucleotide reductase.78 8 0 Instead, a diiron(III) oxygen radical species was invoked as the species which causes the oxidation and deprotonation of the tyrosyl residue. A similar compound has been suggested for Q,34 but such a species would be expected to be paramagnetic. In addition, the O-H bond of tyrosine is significantly weaker than the C-H bond in methane, and the active abstracting species in R2 may not be strong enough to break the C-H bond in methane. It has been speculated that a cysteinyl radical helpts to homolyze this bond.34 If such a species were to couple magnetically with the oxyl radical in Q, it could account for the observed diamagnetism. In the M. trichosporiumOB3b system, a third intermediate, T, with ,nmaxat 325 nm (£ = 6000 M-1cm-1) was observed in the presence of the substrate 35 nitrobenzene. 68 This species was assigned as the product, 4-nitrophenol, bound to the dinuclear iron site, and its absorption was attributed primarily to the 4- nitrophenol moiety. No analogous intermediate was found with the M. capsulatus (Bath) system in the presence of nitrobenzene. A proposed cycle49 for hydroxylation by MMO is illustrated in Figure 4. Substrate first binds to the complete system containing all three protein components. Addition of NADH next effects two-electron reduction of the hydroxylase from the oxidized Fe(III)Fe(III)to the fully reduced Fe(II)Fe(II) form, bypassing the inactive Fe(II)Fe(III) state. The fully reduced hydroxylase then reacts with dioxygen in a two-electron step to form intermediate L, a diiron(III) peroxide complex. The possibility that L itself is sufficiently activated to carry out the hydroxylation reaction cannot be ruled out. Intermediate L is then converted to Q as shown in Figure 3. Substrate reacts with Q, and product is released with concomitant formation of the diiron(III) form of the hydroxylase which enters another cycle in the catalysis. Reaction of Q with Substrate Figure 5 presents possible mechanisms for substrate hydroxylation by intermediate Q with the assumption that this species is a diiron(IV) oxo complex. In mechanism. (A), direct insertion of the oxygen atom of Q into a C-H bond occurs. Concerted 2 + 2 cycloaddition of the C-H bond to Q to form a metal- carbon bond followed by reductive elimination of the alcohol is outlined in mechanism (B), whereas (C) illustrates heterolysis of the C-H bond by Q followed by recombination to afford product In C(1), a proton is formed and in C(2) a carbocation is generated. Mechansim (D) involves homolysis of the C-H bond by Q followed by return of the hydroxyl group from iron to the alkyl radical. This process is analogous to the oxygen rebound step in the postulated hydroxylation mechanism of cytochrome P-450. Finally, mechanism (E) 36 illustrates abstraction of a hydrogen atom from the substrate by hydroxyl or another radical within the active site to form an alkyl radical which then adds to the iron-bound oxygen atom of Q. An alternative mechanism in which L reacts in a concerted fashion with substrate via electrophilic attack to form a carbon- oxygen bond followed by release of product is outlined in mechanism (F). In F, the 0-0 bond does not cleave the C-H bond. The catalytic hydroxylation mechanism for MMO has been frequently compared to that of its heme analog, cytochrome P-450.856 81 A widely accepted mechanism for P-450 hydroxylations invokes a high valent iron oxo, or ferryl, species which is then postulated to abstract a hydrogen atom from bound substrate 7 7 in a manner similar to that outlined in Figure 5D. The resulting substrate radical can then recombine with the iron-bound hydroxyl radical in the rebound step. Evidence for the presence of substrate radical intermediates in cytochrome P-450 includes studies with radical clock substrate probes from which the rebound rate constant was calculated to be 2 x 1010 s-1.82' 83 Such substrates rearrange rapidly upon the abstraction of a hydrogen atom in the enzyme active site, affording skeletally modified hydroxylation products.8 3 -85 The presence of such rearranged products is taken as evidence that a substrate radical intermediate participates in the mechanism. Studies of MMO from M. capsulatus (Bath) and M. trichosporium OB3b using several radical clock substrate probes, including two much faster probes than those reported previously for experiments with cytochrome P-450, were reported.5 7 For M. capsulatus (Bath) MMO no products consistent with the formation of a substrate radical were detected. This result implied that reactions with those substrates did not generate substrate radical or carbocation intermediates, excluding pathways D 37 and E in Figure 5. Alternatively, any rebound reaction occurring would have a rate constant > 1013 s-1 . Using an identical radical clock substrate probe which did not rearrange upon hydroxylation with M. capsulatus (Bath), rearranged product was detected with MMO from M. trichosporium OB3b.5 7 From the ratio of unrearranged to rearranged products, a rebound rate constant was calculated to be = 6 x 1012s -1 at 30 C for this system. A separate study with another radical clock substrate probe with MMO from M. trichosporiumOB3b reported products consistent with both radical and cationic substrate intermediates.86 Evidence in support of radical intermediates with MMO from both M. capsulatus (Bath) and M. trichosporium OB3b was reported from experiments in which substrate radicals were trapped during turnover. 8 7 88 The amount of trapped radical was not quantitated in these experiments, however. In other reports, no diffusable radical species were detected in reactions with MMO from M. trichosporiumOB3b. 59

With M. trichosporium OB3b, epimerization with exo, exo, exo, exo-2,3,5,6- 8 1 d4 -norbornane upon hydroxylation occurs, which parallels results for cytochrome P-450 hydroxylation with this substrate.8 9 The extent of epimerization with MMO, however, was significantly less, being 2% following hydrogen atom abstraction from the endo position compared to 18% with cy- tochrome P-450, and 5% after abstraction at the exo position as compared to 14% 90 with cytochrome P-450. Allylic rearrangements with 3,3,6,6-d4-cyclohexene occurred in 20% of the MMO hydroxylation products compared to 33% for cytochrome P-450. These two experiments suggest that, with M. trichosporium OB3b, a rebound reaction must occur with a greater rate constant that with cytochrome P-450, in accord with the radical clock substrate work. 38 Since work with the radical clock substrate probes indicated important differences in the hydroxylation mechansims for M. capsulatus (Bath) and M. trichosporiumOB3b, work with (R) and (S)-[1-2H,1-3H]ethane with both enzymes was carried out.91' 92 With M. trichosporium OB3b, approximately 65% of the products displayed retention of stereochemistry.9 1 A rebound rate constant of 3 x 1012 s-1 was calculated, assuming a free energy change of 0.5 kcal mole-1 for rotation about the C-C bond.92 This value is in good agreement with that obtained from the radical clock substrate probe analysis.57 Preliminary experiments with the M. capsulatus(Bath) system indicate that this MMO is prima facia more complex, with the apparent enantioselectivity of the reaction being dependent upon whether hydroxylation occurs at the deuterium atom or at the hydrogen atom of the chiral carbon.92 In some cases, > 90 % retention of the stereochemical center was observed. An enantioselective exchange mechanism in which the alcohol product loses the tritium label was invoked to account for the differences in apparent sterechochemistries. Kinetic isotope effects in the hydroxylation reaction have been measured for MMO from both organisms. With both systems, no significant intermolecular kinetic isotope effect was found for most susbstrates,57 93'94 although a value of 62 kH/kD = 12 was reported with CH4 vs CD4. Substantial intramolecular isotope effects (kH/kID = 5) with M. capsulatus (Bath) were found with several substrates.5793 Studies with M. trichosporium OB3b have revealed intramolecular isotope effects of kH/kD = 4 to 5.81,91 These results indicate that C-H bond breaking is not the rate-determining step in the overall enzymatic reaction. The large intramolecular isotope effect does indicate that the hydroxylation step involves a substantial C-H bond-breaking component. The magnitude of the intramolecular isotope effects for both MMO's can be compared to values of ktH/kD = 7-14 seen with cytochrome P-450 hydroxylations. 7 7 95 39 Some fundamental differences between MMO and cytochrome P-450 have emerged. The ability of high concentrations of hydrogen peroxide to effect a shunt pathway for the M. trichosporiumOB3b MMO has been cited as evidence for a hydroxylation mechanism paralleling that of cytochrome P-450.56 It is noteworthy, however, that oxo transfer reagents such as iodosylbenzene, which support catalysis with cytochrome P-450, cannot effect turnover with MMO. In ,addition, the mechanism for olefin epoxidation by MMO from M. trichosporium OB3b8 1 differs from the proposed mechanism for cytochrome P-450.96 With cytochrome P-450, the 1-trans-proton of propylene exchanges with solvent protons during turnover, from which an epoxidation mechanism involving oxametallocycles and iron carbene intermediates was proposed. In the reaction of propylene with MMO, however, no such exchange occurred. In summary, mechanistic studies have revealed intriguing differences between MMO from M. capsulatus(Bath) and MMO from M. trichosporiumOB3b. With M. capsulatus (Bath), radical clock substrate probes indicated either that a substrate radical is not produced or that it reacts with a rate constant >1013 s-1. Reactions with chiral ethane were primafacia quite complex. Further analysis of the data suggested the operation of an enantioselective exchange mechanism. With MMO from M. trichosporium OB3b, radical involvement was suggested from several experiments, and a rebound rate constant of 3 to 6 x 1012 s-1 was calculated for this system. The different behavior between the two MMO systems has several implications. For each case, two reaction pathways may operate in parallel, one involving a substrate radical (Figure 5D), and one not (Figure 5A-C). The degree to which the radical vs. non-radical mechanism is followed may be imposed by the steric requirements of the substrate in the active site. Alternatively, abstraction of a hydrogen atom to form a substrate radical may occur in both 40 cases, but the rate constants for rebound may differ between the two organisms. For example, in the M. capsulatus (Bath) system, the rebound rate constant may be so large that only an extremely small amount of the substrate radical has a sufficient lifetime for rotation about the C-C bond to occur before recombination with the bound hydroxyl group. Conclusions. Physical studies of the hydroxylase have established the structural nature of the diiron core in its three oxidation states, Ho, Hmv, and Hred. Although the active site structure of hydroxylase from M. trichosporium OB3b and M. capsulatus (Bath) are similar, fundamental differences were observed for other aspects of the MMO system. The interactions between the other components, protein B and reductase, vary substantially. More structural information is necessary to understand how each of the components affects the others with respect to its physical properties and role in the hydroxylation mechanism and to reconcile the different properties seen in the two MMO systems. The kinetic behavior of intermediates in the hydroxylation reaction cycle and the physical parameters of intermediate Q appear similar. The reaction of Q with substrate, however, varies. Formation of radical substrates are more obvious with the M. trichosporium (OB3bsystem. In comparison to the cytochrome P-450 system, the hydroxylation mechanism with both MMO systems either has a rebound rate constant which is much larger, and/or it takes place by an alternative pathway. 41 References (1) Hanson, R. S. In Advances in Applied Microbiology Academic Press: New York, 1980; Vol. 26; pp 3. (2) Enhalt, D. H. In Microbial Producationand Utilization of Gases;Schlegel, H. G., Gottschalk, G. and Pfennig, N., Eds.; Goltze Publishers: Gottingen, 1976; pp

13-22.

(3) Enhalt, D.; Schmidt, U. Pure Appl. Geophys. 1978, 116, 452-464.

(4) Dalton, H.; Leak, D. J. In Gas Enzymol.; H. Degn, Ed.; D. Reidel Publishing Co.: London, 1985; pp 169-186.

(5) Burch, R.; Squire, G. D.; Tsang, S. C. J. Chem. Soc., Faraday Trans. I 1989, 85, 3561-3568.

(6) Colby, J.; Stirling, D. I.; Dalton, H. Biochem. J. 1977, 165, 395-402.

(7) Dalton, H. In Adv. Appl. Microbiol. Academic Press: 1980; Vol. 26, pp 71-87.

(8) Green, J..;Dalton, H. J. Biol. Chem. 1989, 264, 17698-17703. (9) Adriaens, P. App. Environ. Microbiol. 1994, 60, 1658-1662.

(10) Jahng, D.; Wood, T. K. App. Environ. Microbiol. 1994, 60, 2473-2482.

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o\ o, 0" ON '4 kO\ \I \C \ dt d m m m c t ct 0) 0 o\ o -5 0 LrL Lo r-q rocd CZ I-' z0) z cuE rt Z, C" C * tM 3nZ.- !e O 00 N N M ct to) 0)CZQ ; . . C)E cnk 0)O

tn 00 t 00 .b o

tno Z; \IC n ' N N N N \D 'IC \I \t C4 t- t to to tO N" N N \IC O

N rN _r) CD 0N O 00 0U eN r

UO ·k ©)0 0I.-,4 0FU .Z1.;I -:z£0° ON C, m C 00 00N -. --- . . . I I ti Vo

=rz

- :r. -oh ,.Q :,.o d cn °..: a'"E ao 'z~- tE - (0 C - N .:nrO- nX. E1 r.- E ** E E S N > $; u . E ,E-q (, x E

tc '5; co W0 +CO ; U ) CZ 0 t O E :0 0Z Ei z 49 Table 2. Reduction Potentials (mV vs NHE, pH 7.0) of the MMO Hydroxylase from M. capsulatus (Bath)60,61 and M. trichosporium OB3b. 6 3

° 0 Component(s) Conditionsb E1 E2

H 1 48 ± 5 -135 +5

H, propylene 1 30 ±+5 -156 ± 5

H, protein B, R 1 a a

H, protein B, R, propylene 1 < 100 + 25 = 100 + 25

H 2, 3 100 ± 15 -100 + 15

H, protein B 2, 3 50 ± 15 -170 ± 15

H (M. trich.) 4 76 + 15 21 + 15

H, protein B (M. trich.) 4 -52 + 15 -115 + 15

H, protein B, R (M. trich.) 4 > 50 + 15 > -170 + 15

H, R (M. trich.) 4 > 50 + 15 >-170 + 15

H, protein B, R, propylene 4 > 50 15 > -170 15 (M. trich.) a No reduction was observed in these cases. bSee appropriate references for specific experimental conditions. 50

r.o C I0 0 ~1 o 8 o I m r-. II C) A Lt) d U 2 o 0C. z1-4 C-0 o 0 o r) C7\ 00 O ON c n C.Z 00 C Lr) r V A L-) 4Z.W a. z 0 0 .5 .1 C. rS: Z Z C o L. C or) ad P6 u cj, CS C: C) U uGd ac:~ W u 75 U o a a X CZ x o X Gd3 o 0 o~~~~~~~~ ; 4 o US c =CO'' 7O 4e Xr Gd

gE uo r X03 I.,- U d x(t E o tA O 51 Table 4. Spectroscopic and Kinetic Parameters for Intermediates in the Reaction of Hred with Dioxygen. M. M. trichosporiuma capsulatus (Bath)b

Q L Q

optical kmax, nm 330 (7500) =625 (500) 420 (3600) 430 (7500) 350 (1800) (E, M-lcm - 1 )

kform, S- 1 1 22 0.5

kdecay, 1 - 0.05 0.07 M6ssbauer site 1: 6, mm/s 0.17 0.66 0.21 AEQ, mm/s 0.53 1.51 0.68 site 2: 6, mm/s 0.14 AEQ, mm/s 0.55

kform, s - 1 25 0.3

kdecay, s - 1 0.3 0.03 Raman cm-1 904 aSee refs. 67 and 68. bSee refs. 49, 69, 70. 52

00

C;

U

z

0.)

C 0.) 0.)

U

0. 'S C 0.

Ul

'-4 CI 53

CN

5q el.z NA

,I

NC,,

I I

5- 54

.

cl

I-

0o a)

I-

t-4

-0·.. 55

0 0 I Z z

0 C

tC14 / o -

_ N

I ::.L 0OI\ Z0 0

0 0 I Z Z _©,11 I)-/ 0--~f--_-© 'O

Ittl ,0 0 .0 0I\

oflo 0 I _I Z v O.- L.

z0 0-0 Joz Z 0 Z 56

Figure 3. Proposed route for conversion of intermediate L to intermediate Q. The resulting diiron(IV) oxo species could bind oxygen with one iron, or the oxygen could be bound symmetrically by both iron atoms. 57

'l'

\0 - O-Feo -0 N \ 0 N

2H+

H2 0

0 H O \ jo,0 O-FeV -\. Fe'eIII -0O / \o / N N O O

H O O H O o I 0 OO-- Fe FelV< FeI IV-O o F or / \-O \ N N o0 /00

,Q" 58

Figure 4. Proposed catalytic cycle for the hydroxylation of hydrocarbons by MMO. 59

ROH + H 0 R-H. 2 FeIII FeII'I H -1

R-H R-H J FeII,- Fe"' shunt H pathway

0.31 s -l NADH

+ 2H R-H NA)', -+ Hw ~.lt.T Fe2 III(02 -2) I e' Fe" 1 "Ll"'~.....~25 s'. I R-H: H

02 60

V1 u4- C u U 0 CY o cC

0

0) x 0) 0

Cx u) o,.4 cu o4 ) o o .4-Z

o o Co

'4- 0' C

C o I z .oE a

.- 4 C a) ._ c U a ,C tC S0) C,

0) .4o 5-4 0) o o Ur C U U U

Cd

C U Lta Ca --

=cC 61

,-i

\ of0-/ ,-

\ 0-4 ©-0

Co

0* I CDi z - 0u -a0.O-=

I wo \ .- 0.* O- C0- O.-* + O-=

0-L- t~~~~~e ~ - 4) l - O.,ll

= wo~ .0-0 0-3: . . a-. ... -IQ

0=

wo O W-0U O-=~

b-31

W-4e = cu

U u 62

0-4 0-

0- 0m.. _ 0-_ 0-* w

wo Lb-3: ~~ ~+ ct C40-= C-4 Ie >+E OS

-

- tr

0 _ a; = cc( e3 o-- 4's

0 O- ;> O= 0LL / &-4 I

a) C4Q v

.."0 To=, ,,/A/

-..I 0-=/ 0=: 63

CHAPTER 2.

Redox Properties of the Hydroxylase

Component of Methane Monooxygenase from Methylococcus capsulatus (Bath) -- Effects of Protein B, Reductase, and Substrate 64 Introduction Methane monooxygenase (MMO) from the methanotrophic bacterium Methylococcus capsulatus (Bath) catalyzes the synthesis of methanol from methane and dioxygen, which is the first step in the metabolic pathway of the organism. 1 MMO exhibits an extremely broad substrate specificity, reportedly inserting oxygen into a wide variety of alkanes, alkenes, ethers, and alicyclic, aromatic, and heterocyclic compounds. 2 In addition, MMO from Methylosinus trichosporium OB3b oxidizes haloalkenes, including trichloroethylene (TCE).3 This reactivity parallels that of the well-studied heme analogue, cytochrome P-450,4'5 although the mechanism of dioxygen activation may be more complex. Soluble MMO from both organisms consists of three components, all of which are necessary for activity.6 The hydroxylase component has a Mr of 251 kDa 7 and comprises three subunits in an o2P2Y2 configuration. The hydroxylase is the site of substrate binding and contains up to two non-heme dinuclear iron cores, the number of which depends on conditions of growth, harvesting, and purification. 8 The fully reduced form of the hydroxylase is thought to be responsible for dioxygen activation. 8 The 38.6 kDa reductase contains one FAD and one [2Fe2S] cluster with reduction potentials of -260 and -220 mV vs. NHE, respectively.9 The oxidized form of the antiferromagnetically coupled [2Fe2S] cluster has no EPR signal, whereas the reduced form has a resonance at gav = 1.96.10 The reductase accepts electrons from NADH and subsequently transfers them to the hydroxylase.1 1 The third component, protein B, has a Mr of 15.5 kDa, contains no prosthetic groups and has been proposed to regulate electron transfer between the reductase and hydroxylase. 12 The hydroxylase has been previously characterized, in part by our laboratory, with the aid of X-ray crystallography as well as optical, M6ssbauer, EXAFS, EPR, and ENDOR spectroscopy. 13- 17 The native oxidized Fe(III)Fe(III) form is EPR-silent due to antiferromagnetic coupling in the bridged diiron core, whereas the mixed valent 65 Fe(II)Fe(III) species exhibits an EPR signal with gav = 1.83 indicative of a non-heme, bridged diiron center (Figure 1). The fully reduced Fe(II)Fe(II) form gives rise to a signal at g = 15 characteristic of its integer spin (Figure 2). The EPR spectra of the protein in all three oxidation states usually contain a minor signal at g = 4.3 assigned to rhombic high spin Fe(III) and a free radical signal at g = 2.01. Each of these signals generally account for less than 5% of total iron present in the sample and arise from trace levels of mononuclear iron and protein associated radical, respectively. The optical spectrum is essentially featureless above 300 nm in any oxidation state. The dinuclear iron core in MMO has features in common with those in other iron oxo proteins such as hemerythrin, ribonucleotide reductase, and purple acid phosphatase. Accurate models for the structural, physical, and chemical properties of the diiron centers in these proteins have been a focus of attention in our laboratory. 18-2 0 The iron core in each protein functions differently. In hemerythrin the iron center reversibly binds dioxygen, in purple acid phosphatase it hydrolyzes phosphate esters, in ribonucleotide reductase it is required to generate a tyrosyl radical, whereas in MMO it catalyzes the selective oxidation of methane to methanol. A compelling question yet to be answered from studies with both the models and the proteins themselves is just how nature has selectively tuned each active site to carry out its particular function. The highly specialized reactivity most likely results from modifications in the coordination environment of the bridged pair of iron atoms. An early study of the hydroxylase reduction potentials reported a large separation between the two reduction potentials separating the Fe(III)Fe(III), Fe(]:I)Fe(III), and Fe(II)Fe(II) states, which suggested that high yields of the mixed- valent form could be produced.2 1 In practice, however, only = 25 % of this state was observed. 14 To reconcile this behavior, the reduction potentials of the hydroxylase were reinvestigated by monitoring the EPR spectrum of the mixed valent form at 66 various potentials. To probe factors that might control the functional properties of the dinuclear iron center, the effects of the reductase and protein B components as well as substrate on the potentials were studied. Moreover, we discovered that addition of substrate to the complex formed by the hydroxylase, protein B and the reductase increased the redox potentials. Most of the work reported here was previously published.2 2 A recent study of the redox properties of hydroxylase from M. trichosporiun OB3b reported different potential values obtained under conditions purported to be closer to the natural system.23 Accordingly, additional titrations were carried out under similar experimental conditions and are reported herein. Experimental Protein Isolation. Growth of M. capsulatus (Bath) and purification of the hydroxylase were carried out as reported elsewhere.14 22 Protein B was prepared as reported.2 4 For experiments with reductase, an unresolved mixture of reductase and protein B obtained from the DEAE cellulose column described in the hydroxylase isolation scheme was used without further purification. Specific activity (propylene) for the hydroxylase ranged from 150 to 250 mU/mg with 2.2 to 2.5 Fe/mole, but all preparations resulted in nearly identical EPR spectra. Redox Titrations. Titrations were performed in a manner similar to the method described, elsewhere,2 1 under one of three slightly varying sets of conditions referred to as A, B and C. Solutions in all conditions typically contained about 5 ml of 100 M hydroxylase in 50 mM MOPS (MOPS = 3-(N-morpholino)propanesulfonic acid, Sigma), pH 7.0. Under conditions A, the following were added (100 jiM each) to mediate electron transfer from the reductant to the hydroxylase: N,N,N',N'- tetramethyl-1,4-phenylenediamine dihydrochloride, 2,3,5,6-tetramethyl-1,4- phenylenediamine, 2,6-dichloroindophenol (sodium salt, hydrate), duroquinone, phenazine methosulfate, thionin, methylene blue, potassium indigotetrasulfonate, 67 Indigo Carmine, anthroquinone-1,5-disulfonic acid (sodium salt hydrate), anthroquinone-2-sulfonic acid (sodium salt, monohydrate), benzyl viologen dichloride (all from Aldrich). The mediators methylene blue and 2,6- dichloroindophenol were omitted in conditions B. Conditions C were identical to B except that the mediator concentrations were lowered to 20 gM. The resulting solution was repeatedly evacuated and back-filled with dioxygen-free argon, then transferred with a gas-tight syringe into an apparatus described elsewhere.25 To perform experiments with substrate present, propylene (Matheson, polymer grade) saturated with buffer was continuously passed through the titration vessel. A redox combination electrode (Pt vs. Ag/AgCl, Corning) was used to monitor the potential. With constant stirring, small volumes of typically 1 to 3 l of a 0.1 M sodium dithionite (Aldrich) solution were used to lower the potential. After a 10 min period, or 90 min for conditions C, determined in time course studies to be sufficient for the system to reach equilibrium, a 300 jgl sample was removed with a gas-tight syringe and then injected into an argon-filled, quartz EPR tube. The tube was immediately frozen in cold isopentane (-140 C) and immersed in liquid N2. For experiments performed with protein B, hydroxylase was incubated with two equivalents of the protein prior to reduction. In studies of the complete system, a mixture of protein B and reductase was added either before addition of reductant or after the fully reduced form of the hydroxylase was produced. The amount of protein B/reductase mixture used was determined by adjusting to achieve maximal hydroxylase activity, each component = 200 M, or twice the concentration of the hydroxylase as estimated by HPLC analysis. EPR Spectroscopy. All EPR spectra were recorded at X-band on a Bruker Model ESP 300 spectrometer with an Oxford Instruments EPR 900 liquid He cryostat set at 9 K. The g < 2 signal was quantitated under non-saturating conditions by 68 double integration of the first derivative spectrum for comparison to a frozen solution of copper perchlorate (mM CuSO4 , 2 M NaC10 4 , 0.01 M HC1). Transition probabilities were corrected for g-value anisotropy. 26 The percentage of mixed valent species was calculated from the concentration of spin relative to the concentration of iron present in the sample. To compare different titrations the mixed valent signals were normalized to the maximum signal observed for each individual titration. Results Hydroxylase Titrations. Three titrations under conditions A were carried out on the hydroxylase component of MMO. Representative EPR spectra are shown in Figure 3. The mixed valent signal increased in intensity as the potential was lowered, reached a maximum at approximately -50 mV vs. NHE, then decreased as the potential was lowered. At roughly -100 mV the fully reduced, g = 15, signal was detected and grew in intensity as the potential was further diminished. The reversibility of the system was confirmed by back titration at several potentials. The maximum intensity of the mixed valent signal corresponded to 85% of the total iron present. This yield most likely reflects the maximum attainable because of the inaccuracies introduced by using a copper standard for the integrations. The reduction potentials of the hydroxylase under all conditions investigated are summarized in Table 1. A plot of the relative intensities of the mixed valent signal versus potential is shown in Figure 4 for conditions A data. Titrations of the hydroxylase were additionally performed under both conditions B and C, and intensities of the mixed- valent EPR signals are plotted in Figure 5. The potentials for stepwise reduction of the hydroxylase diiron(III) core, shown in eq. 1, were obtained by fitting the data in this plot of mixed valent intensities vs. potential to eq. 2. This expression was derived from the two individual Nernst equations governing the reductions and 69 the mass balance equation. The best fit, where n = 1, F is the Faraday constant, R is

E] ° E2° Fe(III)Fe(III) - Fe(II)Fe(III) 2 Fe(II)Fe(II) (1)

0 Fe(II)Fe(III) = 100 / [exp (-nF/RT) (E - E2 )} + exp {(nF/RT) (E - E1°)} + 1 ] (2)

the gas constant, and T = 298 K, yielded potentials of 48 and -135 mV vs NHE for E1° 0 0 and E2 , respectively, with estimated errors of + 5 mV. The E1 and E2 values can also be used to compute the relative amounts of all three redox species present at a given potential. The results of such a calculation are also depicted in Figure 4. The disproportionation constant for the mixed valent species was computed to be 8.04 x 10-4. Fits of the data from titrations under B and C conditions both yielded values of

° ° 100 mV and -100 mV vs. NHE for E1 and E2 , with estimated errors of ± 15 mV, as shown in Figure 5. A plot of the EPR signal intensity versus potential of the mixed valent hydroxylase measured under conditions A in the presence of the substrate propylene as a substrate is also shown in Figure 4. A fit of these data to eq 2 revealed only a slight diminution of the reduction potentials, to values of 30 vs 48 mV and ° ° -156 vs -135 mV for E1 and E2 , respectively. Titrations of hydroxylase with protein ° ° B and were performed under conditions B and C. In these experiments, E1 and E2 were lowered to 50 mV and -170 mV ± 10 mV, also shown in Figure 5. Although shifts in the potential induced by substrate and protein B are small, they do suggest some perturbation of the environment at the diiron center. Component Interactions. Addition of protein B and reductase under conditions A produced a significant change, illustrated in Figure 6A. For all potentials investigated down to -200 mV, no mixed valent nor any fully reduced signal was detected, implying that no reduction occurred. When protein B and 70 reductase were added to the fully reduced form of the hydroxylase, the EPR signal of the latter persisted. A dramatic change resulted when propylene was introduced to all three MMO components. Reduction of the hydroxylase to the diiron(II) state was observed upon initial introduction of reductant, even at potentials as high as 150 mV. Representative spectra are displayed in Figure 6B. Discussion Hydroxylase Potentials. As reported previously,21 the redox potentials for the stepwise reduction of the hydroxylase component of MMO can be determined by monitoring the appearance and disappearance of the g < 2 EPR signal intensity 0 characteristic of the mixed valent form. Our values (El1 = 48 to 100 and E2 = -135 to -100 mV) for the potentials differ from ones reported early2 1 (350 and 25 mV, respectively). We have no obvious explanation for the discrepancy, owing in part to the lack of experimental detail given in the previous study. For example, values for activity and iron content of the hydroxylase were not specified, nor was it clear that the potentials were measured by an electrode. By using the present potential values for the hydroxylase and E°' of 80 mV for phenazine methosulfate we calculate the maximum attainable yield of the mixed valent species at this potential to be 20 to 600%. The redox potentials determined in this study could explain why, at 100 mV, the spin concentration of the M. capsulatus (Bath) hydroxylase was only 0.27 per protein molecule in previous reports.21 Finally, the present results are more in accord with an earlier report for M. capsulatus (Bath) that formation of the mixed valent hydroxylase occurs "at redox values around 0 mV" and that the fully reduced form can be obtained "below - 100 mV".27 Incubation with low concentrations of mediators (20 gM, conditions C vs. 100 pM, conditions B) relative to 100 jgM hydroxylase resulted in nearly identical potentials. A longer time of 90 min, however, was necessary for the solutions to reach equilibrium under for conditions C. This behavior is consistent with the M. 71 trichosporium OB3b work, in which conditions most like set C were employed, and an incubation time of 1 h was used. 0 Although the separation between the potentials (E2° - E1 ) remained the same within experimental error (183 vs. 200 mV) with different mediators present, the potentials changed from 48 to 100 mV and from -135 to -100 mV. The increase of the potentials suggests reduction may be mediator-dependent. A recent report of the determination of M. trichosporium OB3b hydroxylase redox potentials suggested that methylene blue and 2,6-dichloroindophenol may perturb the diiron unit and alter the EPR signal as well as the potentials. We have determined that the lineshape of the signal is not perturbed by addition of these two mediators. The Fe(II)Fe(III) EPR signal in the presence of these two mediators is given in Figure 1, and the signal in their absence is illustrated in Figure 7. No shift in the g-values of the signal was seen, but a small feature at higher field was observed in their absence. Examination of the signal lineshape does not conclusively exclude possible perturbation of the active site by the mediator, making it very difficult to determine which mediator(s), if any, actually do interact with the diiron unit. The increase in potential cannot be explained by loss of the redox buffering capacity in the potential regions covered by the omitted mediators. Table 2 lists all the mediators employed, together with their reduction potentials.2 5 Omission of methylene blue and 2,6- dichlorophenolindophenol removes buffering in the potential ranges of approximately 41 to -19 mV and 247 to 187 mV vs. NHE, respectively, which are not those where the hydroxylase potentials lie. Whereas the reduction does seem mediator-dependent, identifying the nature for the dependence has proven difficult. To combat problems associated with mediators, the reduction potentials could be determined from a set of titrations in which each mediator is systematically omitted, thereby determining the effect of each mediator on the potential. In addition, direct electrochemical measurement of the redox potentials at an electrode surface would 72 eliminate possible complications with mediators, but use of this methodology with proteins has other signficant problems such as protein denaturation and slow rates of electron transfer. These type of experiments were attempted, but no reduction of the hydroxylase was observed.28 ° Literature Comparisons. The E1 and E2 values obtained in this work (100 and -100 mV) differ from the potentials found in studies with hydroxylase from M. trichosporium OB3b (76 and 21 mV) under similar conditions.23 Our values are closer to these numbers than was previously reported (350 and 25 mV).21 The reason for the discrepancy with M. trichosporium OB3b work may be experimental variations, or possibly differences in the MMO systems from the two organisms. Examples of variable behavior between the two systems in other areas include results with radical clock probes,22 unpublished work with chiral ethane as a substrate, and details of the results of kinetic studies of intermediates in the catalytic reaction cycle. Alternatively, the reduction potentials may depend on different mediators employed. In the M. trichosporium OB3b work, two additional mediators were substituted for methylene blue and 2,6-dichloroindophenol, but since these two were not readily attainable, we could not exactly duplicate their experimental conditions. As mentioned above, it appears that the electron transfer mediators are non-innocent, which will complicate comparisons of hydroxylase potential values obtained under different conditions. Structural Implications. Several synthetic model complexes containing diiron oxo and related units are known, but only a few of these display reversible or quasi-reversible redox reactions connecting the Fe(II)Fe(II), Fe(II)Fe(III), and Fe(III)Fe(III) states 29-32 A comparison of the structures and redox properties of the dinuclear iron centers in the proteins and model compounds leads to some interesting observations. Hemerythrin contains an oxo bridge in the fully oxidized state, whereas MMO does not.14 The diiron center in hemerythrin has reduction 73 potentials of El'' = 110 and E2° = 310 mV vs NHE.2 9 Since E1° is less than E2°, the mixed valent form of the protein is unstable with respect to disproportionation to the diiron(II) and diiron(III) forms.33 A mixed valent form of the R2 protein of ribonucleotide reductase, which also contains an oxo-bridged dinuclear iron center, has been observed only in very small quantities (< 10 %).29 The difficulty in ° 0 obtaining the mixed-valent form of R2 suggests that E1 is less than E2 for this form of the protein. The mixed valent form of the oxo-bridged model complex 2+ [Fe2 O(O2CCH 3) 2(Me 3TACN)2] , where Me3TACN is the facially capping ligand 1,4,7-trimethyl--1,4,7-triazacyclonane, is also unstable with respect to disproportionation. 3 0 Thus the oxo-bridged models and proteins may share the ° °. common feature that E1 < E2 The presence of a hydroxo bridge in the oxidized ° 0°. and mixed valent hydroxylase may cause E1 > E2 Binding of reductase and protein ° B could cause significant perturbations shifting E2 > E1 . A diiron complex in which the metals are connected by two phenoxide bridges has been described which undergoes quasi-reversible redox reactions similar to those in eq. 1 with E1 = -9 and E2 = -259 mV vs NHE in dimethylformamide solution. 3 2 Although it is not valid to compare the absolute values of these potentials to those of MMO as determined here, the difference between the two, 250 mV, is reasonably close to the difference between the corresponding protein potentials, 200 mV. In this complex, both iron atoms are octahedrally coordinated to oxygen donor ligands. These observations indicate that the structure of the core has a direct influence on the redox behavior. In particular, an increase in the number of nitrogen ligands relative to oxygen ligands has been correlated to higher reduction potentials of diiron complexes.3 4 The influences of protein B, reductase, and substrate on the hydroxylase reduction potentials could, for example, result from a change in the number or protonation of an oxygen ligand(s). 74 Component Interactions. Work with hydroxylase from M. trichosporium OB3b also indicates that, in the presence of protein B, the g values, and thus the lineshape, of the mixed valent signal change, from 1.94, 1.86, and 1.75 to 1.87, 1.77, and 1.62, respectively.3 5 Protein B additionally caused the reduction potentials of this hydroxylase to shift more negative values, from 76 and 21 mV to -52 and -115 ° 23 mV vs. NHE, respectively, for E1 and E2. For M. capsulatus (Bath) hydroxylase, we have observed a dimunition of the hydroxylase reduction potentials with protein B to values of 50 and -170 mV vs. NHE, as shown in Table 2. In both systems, it is evident that binding protein B must perturb the diiron center, probably by altering the coordination environment of the iron atoms. Additional evidence that protein B alters the coordination sphere of the diiron center is provided by the shifting of hyperfine coupling constants in the ENDOR spectrum of the mixed valent state.36 To elucidate the nature of this perturbation, more structural work of the hydroxylase/protein B complex is imperative. Addition of protein B and the reductase to the oxidized hydroxylase component blocks the mediated transfer of electrons to the dinuclear iron core. Use of NADH as the reductant in place of sodium dithionite yielded similar inhibition, and suggests that the electron transfer pathway through either the mediators or the reductase is the same. These results account for previous observations that a mixture of hydroxylase and reductase oxidizes NADH with no substrate turnover, but that addition of protein B to this mixture results in NADH consumption only when substrate is oxidized.l 2 The same report stated that the rate of NADH oxidation by a mixture of hydroxylase and reductase was independent of substrate and. occurred at only 40% of the rate in the complete system (hydroxylase, protein B, reductase, and substrate) to produce either hydrogen peroxide or water. These observations indicate that protein B is necessary to obtain high electron transfer rates, and to couple the transfer with substrate turnover. Studies of MMO from M. 75 trichosporium OB3b indicate that only a large excess (17 equivalents) of protein B can inhibit mediated reduction of hydroxylase in the absence reductase.3 5 Our observations for the M. capsulatus (Bath) enzyme reveal that mediated electron transfer is completely inhibited upon addition of protein B and reductase at ratios that maximize hydroxylase activity, approximately 2:2:1 molar quantities of reductase:protein B:hydroxylase. These results, together with those from the previous studies, 12 indicate that both protein B and the reductase may be necessary to inhibit efficiently reduction of the hydroxylase. This inhibition could either be thermodynamic, for example by alteration of the iron coordination environment in some way and hence the redox potentials, or kinetic, through blocking access of reductant. The finding that the addition of protein B and reductase to the fully reduced hydroxylase does not lead to reoxidation of the enzyme implies that the inhibition of electron transfer is kinetically controlled. This explanation is attractive in view of evidence that stable complexes form among the three protein components. 35 filtshould be noted that electron transfer from sodium dithionite to the redox mediators is believed to be fast relative to the rate of direct electron transfer to the proteins,2 5 and so the potential changes are indeed buffered. Because we limited our redox titration to potentials greater than - 210 mV (Figure 5), no EPR signal characteristic of the reduced reductase (g = 1.96)was observed. Perhaps the most interesting discovery of the present investigation is that, upon addition of propylene to the three components of MMO, electrons are transferred to the hydroxylase even at the highest potentials monitored, > 150 mV. Electron transfer into the dinuclear iron center of the complete system is thus tightly regulated, being allowed to occur only when substrate is present. Since no mixed 0 ° ° valent is observed, E2 has shifted such that E2 > E1 , which indicates that a change in the coordination sphere of the diiron unit has taken place. E2 is estimated from the amplitude of the diiron(II) signal at varying potentials to be 100 25 mV vs. 76 NHE. This estimate should be considered to be only a rough approximation since the nature of the g = 15 EPR signal makes it difficult to quantitate accurately. These findings are consistent with the steady-state kinetic analysis of the MMO complex reported previously, where electron transfer was observed to occur only after substrate was bound.37 Such an increase in reduction potentials is similar to behavior seen with cytochrome P-450, where small changes in the coordination sphere of the heme iron due to the binding of the substrate can raise the reduction potential from -300 to -170 mV.38 Parallel work with MMO from M. trichosporium OB3b failed to demonstrate a similar mode of regulation.23 Instead, it was reported that the potentials of the hydroxylase/reductase, the hydroxylase/reductase/protein B, and the hydroxylase/reductase/protein B/substrate complexes were "slightly more positive" than the values for hydroxylase alone. These discrepancies may be attributed either to procedural or protein variations. The radically different behavior of the MMO complex in the presence and absence of substrate has several implications. As stated earlier, binding of protein B and reductase prevents reduction of the hydroxylase, probably kinetically. Binding of substrate to the hydroxylase not only reverses this effect, but also renders the hydroxylase more electron deficient and hence easier to reduce. This thermodynamic effect would occur only when all three components are present, and the greater driving force could explain the faster rates of NADH oxidation observed when protein B is available. The increased affinity for electrons of the complete MMO system in the presence of substrate could be due in part to enhanced electrophilicity of the diiron(III) core through proton transfer or hydrogen bond formation involving one or more ligands in the coordination sphere. Since the hydroxylase/protein B/reductase complex3 5 appears to be responsible for the inhibition of electron transfer without substrate, the enhancement with substrate might involve a conformational change at the dinuclear iron center. More 77 structural work involving the component interactions is necessary to reach firm conclusions in this area. Regulation of electron transfer as just described would be beneficial to the organism in many ways. The obvious advantage of the kinetic inhibition is that when no substrate is present, no NADH consumption takes place, thereby preventing the wasteful consumption of reducing equivalents. 12 If reduction were allowed to occur in the absence of substrate, there would be a greater chance that the enzyme might be inactivated through generation of the highly reactive, high energy ° species required for the oxygenation reaction. By moving E2 > E2 , the enzyme prevents formation of the catalytically inactive mixed-valent form. From an energetic standpoint, the shift to higher reduction potentials with substrate present is consistent with the core becoming more electron deficient. This potential shift would increase the reactivity of an activated iron core. Without protein B and reductase, such a species would be lower in energy owing to the lowered reduction potentials. In single turnover experiments in which hydroxylase is chemically reduced to the diiron(II) form, low yields are generally observed in the absence of the other components.3 9 This argument implies that the higher energy activated hydroxylase iron core that is generated in the presence of protein B and reductase may be required for efficient substrate oxidation. 78 References (1) Hanson, R. S. In Advances in Applied Microbiology Academic Press: New York, 1980; Vol. 26; pp 3.

(2) Green, J.; Dalton, H. J. Biol. Chem. 1989, 264, 17698-17703.

(3) Fox, B. G.; Borneman, J. G.; Wackett, L. P.; Lipscomb, J. D. Biochemistry 1990,

29, 6419-6427. (4) Ortiz de Montellano, P. R. Cytochrome P-450 Structure, Mechanism, and Biochemistry; Plenum Publishing Corp.: New York, 1986, pp. 217-271. (5) Dawson, J. H.; Eble, K. S. Adv. Inorg. Bioinorg. Mech. 1986, 4, 2-64.

(6) Fox, B. G.; Lipscomb, J. D. Biochem. Biophys. Res. Comm. 1988, 154, 165-170.

(7) Colby, J.; Dalton, H. Biochem. J. 1978, 171, 461-468.

(8) Fox, B. G.; Froland, W. A.; Dege, J. E.; Lipscomb, J. D. J. Biol. Chem. 1989, 264,

10023-10033.

(9) Lund, J.; Dalton, H. Eur. J. Biochem. 1985, 147, 291-296.

(10) Colby, J.; Dalton, H. Biochem. J. 1979, 177, 903-908.

(11) Green, J.; Dalton, H. Biochem. J. 1989, 259, 167-172.

(12) Green, J.; Dalton, H. J. Biol. Chem. 1985, 260, 15795-15801.

(13) Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J.; Nordlund, P. Nature 1993,

366, 537-543.

(14) Dewitt, J. G.; Bentsen, J. G.; Rosenzweig, A. C.; Hedman, B.; Green, J.; Pilkington, S.; Papaefthymiou, G. C.; Dalton, H.; Hodgson, K. O.; Lippard, S. J. J. Am.

Chem. Soc. 1991, 113, 9219-9235. (15) DeRose, V. J.; Liu, K. E.; Kurtz, D. M., Jr.; Hoffman, B. M.; Lippard, S. J. J. Am.

Chem. Soc. 1993, 115, 6440-6441. (16) Fox, B. G.; Surerus, K. K.; Miinck, E.; Lipscomb, J. D. J. Biol. Chem. 1988, 263, 1053-1056. 79 (17) Hendrich, M. P.; Fox, B. G.; Andersson, K. K.; Debrunner, P. j.; Lipscomb, J. D.

J. Biol. Chem. 1992, 267, 261-269. (18) Lippard, S. J. Angew. Chem. Int. Ed. Engl. 1988, 27, 344-361. (19) Tolman, W. B.; Liu, S.; Bentsen, J. G.; Lippard, S. J. J. Am. Chem. Soc. 1991,

113, 152-164.

(20) Feig, A. L.; Lippard, S. J. Chem. Rev. 1994, 94, 759-805.

(21) Woodland, M. P.; Patil, D. S.; Cammack, R.; Dalton, H. Biochim. Biophys. Acta

1986, 873, 237-242.

(22) Liu, K. E.; Ilippard, S. J. J. Biol. Chem. 1991, 266, 12836-12839, 24859.

(23) Paulsen, K. E.; Liu, Y.; Fox, B. G.; Lipscomb, J. D.; Miinck, E.; Stankovich, M. T.

Biochem. 1994, 33, 713-722.

(24') Liu, K. E.; Johnson, C. C.; Newcomb, M.; Lippard, S. J. J. Am. Chem. Soc. 1992,

115, 939-947.

(25) Dutton, L. P. Methods in Enzymol. 1978, 54, 411-435. (26)1 Asa, R.; Vanngard, T. J. Mag. Res. 1975, 19, 308-315.

(27) Dalton, H.; Leak, D. J. In Gas Enzymol.; H. Degn, Ed.; D. Reidel Publishing Co.:

London, 1985; pp 169-186. (28) This work was attempted in collaboration with Professor Frasier Armstrong at the University of California, Irvine. (29) Que, L., Jr.; True, A. E. Prog. Inorg. Chem. 1990, 38, 97-200.

(30) Hartman, J. A.; Rardin, R. L.; P., C.; Pohl, K.; Nuber, B.; Weiss, J.; Papaefthymiou, G. C.; Frankel, R. B.; Lippard, S. J. J. Am. Chem. Soc. 1987, 109, 7387-

7396.

(31) Snyder, B. S.; Patterson, G. S.; Abrahamson, A. J.; Holm, R. H. J. Am. Chem.

Soc. 1989, 111, 5214-5223. (32) Stassinopoulos, A.; Schulte, G.; Papaefthymiou, G. C.; Caradonna, J. P. J. Am. Chem. Soc. 1991, 113, 8686-8697. 80 (33) Armstrong, F. A.; Harrington, P. C.; Wilikins, R. G. J. Inorg. Biochem. 1983, 18,

83-'91. (34) Rosenzweig, A. C.; Feng, X.; Lippard, S. J. In Applications of Enzyme Biotechnology; Kelly, J. W., Eds.; Plenum Press: New York, 1991. (35) Fox, B. G.; Liu, Y.; Dege, J. E.; Lipscomb, J. D. J. Biol. Chem. 1991, 266, 540-550.

(36') DeRose, V. J.; Liu, K. E.; Lippard, S. J.; Hoffman, B. M. manuscript in preparation. (37) Green, J.; Dalton, H. Biochem. J. 1986, 236, 155-162.

(38) Raag, R.; Poulos, T. L. Biochem. 1989, 28, 917-922. (39) Single turnover experiments reported in Chapter 6 are maximized with two equivalents of protein B, although protein B actually diminished the hydroxylase reduction potentials. In this case, the increase in reactivity could arise from kinetic effects. For example, steric modifications of the active site imposed by protein B could occur which cause the substrate to bind tighter, thereby coupling oxidation of Hred with substrate hydroxylation more efficiently. Reductase did not affect the rate constant or yield of the reaction. Mediated reduction of Hox was inhibited by the substrate of the reaction, nitrobenzene. This limitation prevented the production of

Hred from Hox in the presence of protein B, reductase, and substrate to test the reactivity of this putative highly reactive species. 81

Table 1. Reduction Potentials (mV vs NHE, pH 7.0) of the MMO Hydroxyla.se from M. capsulatus (Bath) Under Various Conditions (H = hydroxylase, R = reductase). Also included are the potentials for M. trichosporium OB3b hydroxylase23 ,b and hemerythrin, 2 9 both sets of which were obtained under other conditions.

Component(s) Conditions E10 E2°

H A 48 ±+5 -135 + 5

H, propylene A 30 ±+5 -156 + 5

H, protein B, R A a a

H, protein B, R, propylene A < 100 + 25 = 100 + 25

H B, C 100 + 15 -100 + 15

H, protein B B, C 50 + 15 -170 ± 15

H (M. trich.) -52 + 15 -115 + 15

H, protein B (M. trich.) -76 + 15 -21 ± 15

H, protein B, R (M. trich.) > 50 15 > -170 + 15

H, R (M. trich.) > 50 + 15 > -170 15

H, protein. B, R, propylene > 50 + 15 >-170 + 15

(A4. trich.)

Hemerythrin 110 310 a No reduction was observed under these conditions. 82

Table 2. Reduction Potentials of the Mediators Employed in the Titrations (pH 7.0).25 Mediator E° (mV vs. NHE) N,N,N',N'-tetra- 276 methylphenylenediamine diaminodurene 260 2,6-dichloroindophenol 217 duroquinone 140 phenazine methosulfate 80 thionin 56 methylene blue 11 potassium indigotetrasulfonate -46 indigodisulfonate -125

anthroquinone-1,5-disulfonate -174

anthroquinone-2'-sulfonate -225 benzyl viologen -358 83 oH

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CHAPTER 3.

Electron Nuclear Double Resonance Studies of

the Dinuclear Iron Center in the Hydroxylase Component of

Methane Monooxygenase from Methylococcus capsulatus (Bath) 98 Introduction Electron nuclear double resonance (ENDOR) spectroscopy is a valuable technique used in the structural characterization of proteins. 1 This method indirectly affords an NMR spectrum of nuclei that interact with the electron spin of a paramagnetic center. Information is provided through the observed ENDOR frequencies, and directly gives the electron-nuclear coupling constants. The transition of the nucleus of interest is not actually observed, but its interaction with the paramagnetic center is detected through a change in the EPR signal intensity. ENDOR is a broad-band method very specific for nuclei that having a hyperfine interaction with the electron spin system being probed. The hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath) is isolated in its native Fe(III)Fe(III) oxidation state (Hox).2'3 Because of antiferromagnetic coupling between the two Fe(III) atoms (J = -32 cm- 1, H-ex = -2J Si S2), this form of the enzyme is EPR silent liquid helium temperatures.2' 3 The mixed-valent Fe(II)Fe(III) oxidation state (Hm,,) is readily accessible by one-electron reduction of the ciinuclear center, and gives rise to a characteristic EPR signal with gay = 1.83.2,3 Further reduction to the fully reduced Fe(II)Fe(II) hydroxylase (Hred) results in a ferromagnetically coupled, even spin system exhibiting an EPR feature at g = 15.2-4 EPR signals at g = 4.3 and 2.0, both of which account for < 5 % of total iron, are present in spectra of all oxidation states and are assigned to adventitious Fe(III) and protein associated free radical, respectively. In collaboration with Professor Brian Hoffman and Dr. Victoria DeRose at Northwestern University, we have applied the ENDOR technique to study the diiron center in Hmv probing the active site under a variety of conditions to complement parallel structural studies of the diiron center. 99 To obtain information about the coordination sphere of the Fe atoms of Hmv, ENDOR spectra of native and treated samples, including D20- exchanged, 15 N-enriched, 57 Fe-enriched, and H2170-exhanged protein, were measured. Hmvrin the presence of protein B was also studied to monitor protein interactions. Effects of the inhibitor DMSO5 on the diiron unit were determined by comparison to ENDOR spectra of untreated samples. Isotopically labelled DMSO was employed to obtain additional information about its binding. ENDOR spectra of samples treated with the substrates CH4 1 and CD4 were also recorded. For comparison, the H ENDOR spectrum of Hmv from Methylosinuls trichosporium OB3b was collected. Basics of EPR and ENDOR Spectroscopy. 6 '7 An unpaired electron is characterized by a quantum number ms and a magnetic moment, R-e,which are related by equation (1) (g = 2.0023 for a free electron, = electronic Bohr magneton = 0.927 x 10-20 erg G-1). The energy of the electron magnetic quantum levels when a magnetic field H is applied are given in equation (2). Two levels can arise where ms = +1/2, as illustrated in Figure 1. Transitions between the two levels are governed by the selection rule Ams = +1 and occur upon absorption of microwaves according to equation (3), where h = Planck's constant and u = frequency. With the application of a constant microwave

ge = -g I ms (1) E =- e H = g Pm H (2) hu =g H (3) frequency, absorption will occur at a characteristic magnetic field strength. By scanning the magnetic field under these conditions and detecting microwave power absorption, an EPR spectrum is obtained. Saturation of the EPR signal 100 can be achieved at a static magnetic field. The populations of the two energy levels can thus become equalized, thereby causing the absorption of microwaves to cease. The interaction of a nuclear spin with an unpaired electron is next described. The interaction energy in this case contains three terms, shown in equation (4). Division of eq. (4) by Planck's constant, h, yields equation (5). The nuclear Larmor frequency, n, and the electron resonant frequency, ')e, are defined in eqs. (6) and (7). Substitution of t'n and ue into eq. (5) leads to eq. (8), which defines additional splitting of the electronic energy levels as illustrated in Figure 2. The magnitude of the parameter A 1/2 relative to Un

E=g3 H m -gnn H m+hAms m (4)

E/h = (g H ms) / h - (gn En H mI) / h + A ms mI (5)

Un = (gn Pn H) / h (6)

Ue = (g P H) / h (7) E/h = tems - Un mI + A ms mI (8)

can vary. The case where Vn > A 1/2 is shown in Figure 2A, and the reverse,

'un < A 1/2, is illustrated in Figure 2B. EPR transitions (selection rules: Ams = +1, AmI = 0) occur between the energy levels 1 to 3 and 2 to 4 in Figure 2A and between 1 to 4 and 2 to 3 in Figure 2B. The resulting spectrum in both cases will display microwave absorption at two field values. The ENDOR spectrum for the case in Figure 2A is obtained and analyzed as follows. Saturation the EPR signal involving transitions between levels 1 and 3 will result in equal populations of those two energy levels. A second radiofrequency pulse, referred to as the nuclear frequency, is then applied to the system. The nuclear frequency is scanned with the goal of 101 matching n + IA1/2. At the nuclear frequency n + IA /2, for example, energy is absorbed, flipping the nuclear spin mi (Ams = 0, AmI = + 1 for I = 1/2) and causing a transition from level 3 to level 4 to occur. Since the population of level 3 is now less than the population of level 1, electron transitions between level 1 and level 3 resume with the absorption of the microwave frequency, and an increase in the EPR signal is observed. This change in EPR signal intensity is termed the ENDOR effect. In Figure 3A, ENDOR resonances are "Larmor-centered" and expected to occur at two nuclear frequencies, n ± IA 1/2. In Figure 3B the ENDOR spectrum is "hyperfine- centered," with resonances at A 1/2 + 'n. In practice, only one resonance of this doublet might be observed. Experimental Native Samples. Growth of native M. capsulatus (Bath) and purfication of hydroxylase and protein B were carried out as described elsewhere. 2' 8 Specific activites and iron content were in the ranges reported. Growth of M. trichosporium OB3b and purification of its hydroxylase were carried out as reported. 9 Its specific activity for conversion of propylene to propylene oxide was monitored with the use of protein B and reductase from M. capsulatus (Bath). Cross-reactivity between the two systems has been previously reported. 1 0 ll The measured activity was 180 mU/mg of protein, a value considerably less than expected,9 and could be due to the use of protein B and reductase from a different organism. Reactions of M. trichosporium OB3b hydroxylase with hydrogen peroxide produced product amounts in the reported range. 12 The measured iron content, 2.2 mol Fe/mol hydroxylase, was lower than published values,9 for which we have no obvious explanation. 102 15N Enrichment. M. capsulatus (Bath) cells were grown in an 15N enriched medium 13 in order to obtain correspondingly 15 N-enriched hydroxylase samples. The usual media for fermentations was altered only in 15 that KNO3 (99 % N, Cambridge Isotope Laboratories) was used to inoculate five 5 ml cultures with cells from frozen stocks. The head space in each tube was then replaced with methane. The cultures were allowed to incubate at 45 °C and 250 rpm for 4 days. The methane was replaced daily. Once the cultures were opaque, they were used to inoculate five 50 ml cultures. The head spaces of these cultures were again replaced with methane. The 50 ml cultures were allowed to incubate overnight and subsequently used to inoculate the fermentor. The total volume of the fermentation was 10 1.

After 48 hours (O.D. at 540 nm = 8.9) the cells were centrifuged and 51 g of cell paste was collected. Half of this material was then cracked and purified by the usual methods, yielding 247 mg of hydroxylase (2.4 Fe/mole, SPA 190 mU/mg). 57Fe Enrichment. This growth was performed in the general manner described above except that the normal medium 13 was changed such that the source of Fe was Na5 7 FeEDTA, which was prepared as follows. 2 5 7Fe foil (7 mg, 0.125 mmole, Cambridge Isotope Labs) was dissolved in 1 M of ultrapure

HNO 3. Next, 0.5 ml of H20 and 80 mg (0.2 mmole) of Na4EDTA2H 2O were added. After stirring for 5 min, 4.1 mg (0.1 mmole) of NaOH was added. The resulting mixture was allowed to stir for 6 hours, after which it was centrifuged to pellet the precipitate. The dark green supernatant (1 ml) was added to 100 ml of 20 mM sodium phosphate buffer (pH 7.0) and autoclaved as required in the normal medium preparation for fermentation. A 10 1 fermentation was carried out starting from the frozen stocks as described above. The yield of cell paste was 50.3 g. Two 25 g batches of cells were 103 cracked to yield a total of 120 mg hydroxylase (2.0 Fe/mole, SPA = 163 mU/mg).

D20 Exchange. Deuterium exchange of the hydroxylase was achieved in the following manner. Liquid chromatography was carried out at 4 C with a flow rate of 1 ml/min, and the buffer system was 25 mM MOPS (MOPS = N- morpholinoporopane sulfonic acid) in D20, pD 7.0 (as measured with a pH electrode) with 50 mM NaCl unless otherwise specified. Purified hydroxylase (= 50 mg) was loaded on a 1.5 x 5 cm Q Sepharose anion exchange column that was previously equilibrated with 25 ml buffer. The bound protein was next washed for 30 min and the wash buffer was discarded. Fresh buffer (100 ml) was circulated over the column for either 8 or 15 hours. The hydroxylase was then eluted with buffer containing 0.3 M NaC1. 17 H2 0 Exchange. Since this reagent is quite expensive ($300/ 1 ml, 20 % enriched, Isotech), the D20 procedure was not followed. Instead, hydroxylase (= 50 mg) was concentrated to about 100 l and then diluted with 1 ml of 17 HE2 0. The resulting mixture was allowed to incubate for at least 12 hours before concentrating the sample for preparation of Hmv. Sample Preparation. Hydroxylase, 50 mg per sample, was first thawed and then concentrated to a volume of 2 ml at 4000 rpm using centriprep centrifugal concentrators with a molecular weight cut-off of 40,000. The 2 ml concentrates were then placed in centricon concentrators with the same molecular weight cut-off. The sample volume was decreased to 100 g1l, resulting in a concentration of at least 1 mM. A small volume of a mediator stock solution that contained phenazine methosulfate, methylene blue, and potassium indigotetrasulfonate (10 mM in each, dissolved in H20) was added to achieve a :L mM concentration of each mediator in the sample. The solution was placed in a vial, sealed with a septum, and then made anaerobic 104 by repeatedly evacuating and back-filling with dioxygen-free argon. To reduce each batch, a small volume of a 0.1 M solution of sodium dithionite was added to achieve a concentration of = 1 mM sodium dithionite in the sample. The solutions were allowed to equilibrate for 30 min, and at the same time were brought into anaerobic chamber (Vacuum Atmospheres). The samples were then loaded into the appropriate tubes, capped with a septum, brought out of the box, and immediately frozen in liquid N 2. All tubes were stored in the liquid N2 refrigerator before being sent out on dry ice to Northwestern University for ENDOR spectral studies. Addition of Protein B. Protein B was added to the hydroxylase either before the addition of reductant, or after equilibration of the hydroxylase with sodium dithionite. Samples were made that had from 2 to 4 equivalents of protein B (3.8 to 7.6 mg, 2.2 x 10- 7 to 4.4 x 10-7 mole) relative to hydroxylase (50 mg, 2.0 x 10- 7 mole). Addition of Inhibitors/Substrates. Dimethylsulfoxide, DMSO, was added before the sample solution was made anaerobic. A concentration range of 50 mM to 0.2 M of DMSO was studied to monitor ENDOR spectral changes for native Hm, to fully bound Hmv + DMSO. Treatment of the protein with 13 isotopically enriched DMSO (d6-DMSO, 98 %, Cambridge isotope Labs; C- DMSO, 98 %, Isotech) was carried out in an identical manner.

Samples that contained CD4 or CH4 were prepared as follows. The appropriate gas was syringed through the septum of the vial containing the protein solution in place of argon during the last two cycles of evacuation and back-filling. The reaction mixtures were allowed to equilibrate for 5 min before sodium dithionite was added to initiate the reduction of the protein. After this point, the procedures for incubating the sample and loading and freezing the quartz ENDOR tubes were identical to the ones outlined above. 105 ENDOR Spectroscopy. Continuous wave (CW) ENDOR spectra were recorded by Dr. DeRose at Q band (35 GHz microwave frequency) on a locally designed instrument. Through evacuation of the liquid helium cryostat headspace, temperatures were maintained at 2 K. The dispersion mode EPR signal was detected with 100 kHz magnetic field modulation. Results and Discussion EPR Spectrum. The dispersion-detected rapid-passage EPR signal of Hmv is given in Figure 4. The spectrum was simulated with the g values of gi

= 1.94, g2 = 1.87, and g3 = 1.74, and displays some heterogeneity around g2. At Q band, g strain is enhanced which prevents the transition at g3 from being resolved. In a previous EPR report of Hmv from M. trichosporium OB3b, a signal with g values of 1.98, 1.87, and 1.70 corresponding to = 25 % of the total signal was detected.5 This component, thought to be the result of contamination from protein B, was not observed in our studies. The EPR spectrum of Hmv + DMSO is given in Figure 5 along with a simulation. The EPR signal of Hmv in the presence of DMSO is sharpened, which agrees with a previous report of Hmv from M. trichosporium OB3b.5' 14 Some heterogeneity was observed in the sample, however. The simulation in Figure 5 was created from two species, corresponding to 75 % and 25 % of the sample signal. The major species displays g values of 1.95, 1.86, and the minor component exhibits g vaues of 1.93, 1.84, and 1.82. 1H ENDOR Spectra. Three classes of proton resonances are observed for Hmv, as illustrated in Figure 6, depending on the strength of their hyperfine couplings. The spectrum in Figure 7A partially resolves at least seven weakly coupled doublets with A < 4 mHz (see Table 1). Most of the resonances persist in a D20 exchanged sample, shown in Figure 7B, and are isotropic, meaning that A varies little at different g values. This class 106 represents protons that are probably two or more bond lengths away from the diiron center. The second class exhibits intermediate strength coupling constants of A = 7.8 mHz which are highly isotropic. This group of protons is most likely solvent-derived since the intensity of the resonances decreases dramatically after exchange with D20, shown in Figure 6. These properties resemble the previously observed behavior of H20 and OH- bound to the [4Fe-4S] cluster of aconitase.15 Accordingly, these protons were assigned to a 16 terminal H20 or OH- ligand on one of the Fe atoms.

The third class of protons are also exchangeable with D20, but have strong, anisotropic coupling constants. At gl, a doublet with A = 14 MHz is visible, as denoted by the brace in Figure 6. Increasing the magnetic field causes the signal to split into three doublets, marked by the bracket in Figure 6. Maximal coupling is detected at g2 with A 30 MHz. These features were simulated as arising from a single proton, the resonances of which display hyperfine anisotropy at field values away from the edges of the EPR envelope. 'The unusual properties of this proton class do not match behavior expected for an exchangeable proton from a terminal ligand,16 such as histidine, nor do they resemble characteristics of a group hydrogen-bonded to an Fe ligand.17 1H ENDOR of semimet azidohemerythrin contains a similarly behaved resonance. 16 It is widely accpeted that Hr contains a OH- bridge in the semimet oxidation state.1 8-21 We therefore assigned the third set of resonances in Hmv as arising from the hydrogen atom of a bridging OH- ligand. Hmv samples from M. trichopsorium OB3b displayed very similar resonances in the 1H ENDOR, and the same assignment of the coordination environment of the Fe atoms was made.1 6 Subsequent ESEEM studies with M. trichopsorium OB3b confirmed this work.2 2 Analyses from the X-ray 107 crystal structure,2 3 and both EXAFS and electronic spectroscopy2 of Hox from M. capsulatus (Bath) are consistent with the 1H ENDOR assignments for Hmv. Figure 8 shows an ENDOR spectrum of Hmv in the presence of DMSO. Some differences from the Hmv signals shown in Figure 5 were detected. The resonances overall are sharper, and the A values of some were shifted, as reported in Table 1. The signals of the terminal H20 ligand and the OH- bridge persist in the presence of DMSO, as shown in Figure 8, indicating that these ligands remain bound. 1 4 N and 1 5N ENDOR Spectra. Figure 9 displays the 14 N and 1 5 N

ENDOR spectra of Hmv at g = 1.94. The 1 5N resonances are easier to assign since they lack quadrupolar splitting by the 14N nucleus. Accordingly, 15N data were analyzed first, which facilitated assignment of the more complex patterns in the 14N spectra. Table 2 lists all of the hyperfine coupling constants extracted from the spectra. Four peaks at = 5.5, 9.0, 14.0, and 18.1 MHz are detected in the 15N

ZENDOR spectrum as illustrated in Figure 9B. The resonances at 5.5 and 14 MHz are attributed to remote 15N and 13C nuclei that are coupled to the electron spin by the "distant ENDOR effect" and resonate at their Larmor frequency.24 2 5 The signal at 18.1 MHz corresponds to the u+ (+ = A/2 + vn) peak of a hyperfine-centered doublet with A = 25 MHz (A/2 = 12.5 MHz, n = 5.6 MHz), and the corresponding nitrogen atom is referred to as N1. The more weakly coupled nitrogen (N2) at 9.0 MHz iss assigned as the u+ resonance of a Larmor-centered doublet with and A = 6.8 MHz. The ESEEM signals from both the coordinated and non-coordinated nitrogen atoms from a histine ligand were previously reported.2 6 In accord with that work and other structural data, N1 and N2 are interpreted as histidine nitrogen atoms, one coordinated to each Fe atom. The hyperfine coupling of a nitrogen atom 108 to the Fe(III) site of Hmv is predicted to have twice the magnitude of the coupling to a Fe(II) site based on spin exchange interactions.2 7 N1 and N2 are accordingly assigned to coordinate the Fe(III) and Fe(II) sites, respectively. Figure 10OBillustrates the spectrum of an 15N Hmv sample that was treated with DMSO. Unlike the spectrum of the untreated sample, five resonances are distinguished. The signals present at 5.5 and 14 MHz are attributed to 15N and 13C nuclei, respectively. The resonance at 9.4 MHz is the u+ peak of N2, with A = 7.8 HHz. The two remaining signals, at 15.5 and 17.4 MHz, are both assinged to N1. The peak at 17.4 MHz is the u)+transition of a hyperfine-centered doublet with A = 23.8 MHz, and the feature at 15.5 is a Larmor-centered doublet with A = 20.0 MHz (A/2 = 10.0 MHz). These two new signals, identified as Nla and Nib, correspond to nitrogen coordinated to the Fe(III) site in Hmv. The two hyperfine coupling values probably arise from two distinct species, in accord with the sample heterogeneity seen in the EPR signal. DMSO appears to shift the hyperfine coupling of N2 only slightly, from 6.8 to 7.8 MHz, whereas it signficantly alters the coupling of N1 coordinated to the diiron unit. This behavior differs dramatically from that derived from a previous ENDOR spectroscopic study of Hmvxfrom M. trichosporium (O)B3bin which addition of DMSO left N1 unchanged and N2 was reported to dissociate from the iron center.5 The 14N ENDOR spectrum of Hmv is given in Figure 9A, and was assigned with the aid of the 15N data interpretations. The 14N and 15N resonances are related by their nuclear moments according to un( 15N)/,n( 14N) = A( 15 N)/A(14N) = 1.4.6 Values of 5.6 MHz for un(15N), 25 MHz for A(15 N1) and 7 MHz for A(1 5 N2) correspond to un(14 N) = 4 MHz, A(1 4N1) = 18 MHz and A(14N2) = 5 MHz. As mentioned earlier, the nuclear quadrupole interaction

(3P) of 14N further splits the spectrum and leads to u+ = A/2 + un ± 3P/2. The 109 resonances at 12 and 14 MHz thus arise from N1 with 3P = 2 MHz. The N2 ligand displays signals at 7 and 9 Mhz, with 3P = 1.9 MHz. In the presence of DMSO (Figure 10A) the u+ peak of Nla is predicted to have A = 16.9 MHz. The resonances at 12.1 and 13 MHz were therefore assigned to Nla with 3P(Nla) = 0.9 MHz. Similarly, the u+ transition of Nib is expected to occur at 11.1 + 3P/2 MHz. Setting 3P = 1.9 MHz allowed the resonances at 10.2 and 12.1 MHz to be attributed to N1b. For N2, A(15N) = 5.5 MHz was observed, and A(14N) = 6.7 is calculated. Transitions for o+ are then predicted to be at 6.7 ± 3P/2 for the 14N spectra. Two possible sets of resonances in Figure 10A satisfy these criteria. A value of 3P = 3.7 MHz predicts that the set at 5 and 8.6 MHz correspond to N2. Alternative assignments for N2 are the resonances at 6.8 and 5.5 MHz, with 3P = 1.3 MHz. 57 Fe ENDOR Spectra. 5 7Fe ENDOR spectra of Hmv with and without added DMSO are given in Figures 11 and 12, respectively. At gl, two sets of Larmor-centered resonances are observed. Hyperfine coupling values for Fel, A = 60 MHz (Figure 11A) and Fe2, A= 38 MHz (Figure 11A), were determined. Fel displayed isotropic hyperfine coupling across the EPR envelope, whereas Fe2 exhibited highly anisotropic behavior. Analysis of the spin exchange coupling between the Fe(III) and Fe(II) spins of the diiron center leads to the prediction that: Fel is the Fe(III) center and Fe2 the Fe(II) center. 2 7 Addition of DMSO did not significantly perturb the hyperfine parameters of either Fel or Fe2, as seen in Figures 11B and 12B. Analysis of the 57Fe data is thus consistent with that reported for Mbssbauer and ENDOR studies of 57Fe Hmv from M. trichosporium OB3b.1 4 Samples with Isotopically-Labeled DMSO. ENDOR spectra of samples 13 of Hmv and DMSO labelled at the methyl groups, either d6- or C-DMSO, are illustrated in Figure 13. Both samples display Larmor-centered peaks, but 110 neither spectrum is sufficiently resolved for easy interpretation because the coupling is very weak. When nuclei are weakly coupled, Mims pulsed ENDOR can be used instead of continuous wave ENDOR to resolve the resonances. 24 Figure 14 contains such spectra of the labelled samples. The

resonances are Larmor-centered with A - 1.2 MHz for 13C and A - 0.8 MHz for 2H. Simulation of electron spin echo envelope modulation (ESEEM) spectra can be used to quantitate the number of interacting deuterons.24 The d6-DMSO Hmv, sample displays modulation in its 3-pulse ESEEM spectrum from the substrate molecule, as shown in Figure 15A. The spectra in Figures 15B and 15C were simulated with contributions from three and six deuterons, respectively. The amplitude pattern in Figure 15C does not match that of the experimental spectrum. The Fourier transforms of spectra corresponding to the ESEEM spectra in Figure 15A - C are given in Figures 15D - F. Spectra in Figures 15E and 15F illustrate that amplitudes in the second and third harmonics of the deuteron frequency increase with contributions from a greater number of deuterium nuclei. Since the simulation in which three deuterons contribute exhibits properties most similar to the sample spectrum, it was concluded that only one methyl group of DMSO is coupled to the diiron center. This result indicates that DMSO is bound in an asymmetric manner with respect to the Fe atoms. This behavior is consistent with the observation from 15N ENDOR spectrum that DMSO signficantly affects the coordination environment of the Fe(III) atom. The methyl group is most likely interacting with the Fe(III) atom of Hmv.

Other Samples. The H20 and OH- ligands are both predicted to be 17 exchangeable, but no signals have yet been observed with H 2 0 exchanged samples. It is possible that exchange is extremely slow, and the conditions 111 under which it was performed were not stringent enough. A higher level of 17 enrichment of H 2 0 (> 20 %) may also facilitate the observation of an 170 resonance. Similarly, no new signals were observed with the substrates CD4 or CH 4 added to Hmv. The EPR spectrum of Hmv is altered in the presence of protein B, and ENDOR analysis of these samples is in progress. ESEEM methods to study Hred have been developed using the 14N and 15N samples described in this report. 28 Conclusions ENDOR spectroscopy has provided valuable structural information 1 about Hmv. From H couplings, a terminal H20 ligand and a bridging OH- group were identified. Hyperfine coupling constants of the nitrogen atoms from the imidazole ligands and the Fe atoms have been determined. The effects of the inhibitor DMSO on the characteristics of the ligands bound to the diiron center have been studied, and information about how DMSO binds has been obtained. These studies provide a method to investigate the interactions with other substrates to map the binding site of Hmv. This information will be useful in correlating structural studies of the enzyme with mechanistic information about the hydroxylation reaction. 112 References

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(23) Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J.; Nordlund, P. Nature 1993, 366, 537-543. (24) Biological Magnetic Resonance, Volume 13: EMR of Paramagnetic Molecules; Hoffman, B. M.; DeRose, V. J.; Doan, P. E.; Gurbiel, R. J.; Houseman, A. L. P.; Telser, J., Ed.; Plenum Press: New York, 1993, p 151-218.

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Table 1. 1H Hyperfine Coupling Constants for Hmv and Hmv with DMSO. Type of Proton Aobs(MHz) Aobs (MHz) at gl with DMSO weakly coupled 0.2 0.2 0.4 0.8 0.9 1.4 1.2 1.6 1.8 3.0 2.3 3.4 2.5 3.4 intermediately coupled 7.4 6.0 7.0 strongly coupled 14, 30, 24 (gl, g2, g3) 14, 30, 24 (gl, g2, g3)

Table 2. 14 N and 15 N Coupling Constants of Hmv With and Without DMSO. Values for 14N are predicted from 15N data. Nitrogen Atom Aobs (MHz) at gl Aobs (MHz) at gl with DMSO 15 N1 25 23.8 (NIa) 20.0 (Nlb) 15N2 7 7.8 14 N1 17.9 16.9 (Nla) 14.3 (Nlb) 1 4N2 5 5.6 116

Figure 1. Energy level diagram of a free electron in an applied magnetic field. Energy is the verical axis and magnetic field is the horizontal axis. 117

- ms = + 1/2 (1) Ii?

h = g H

- ms = - 1/2 (2)

Magnetic Field 118

Figure 2. Energy level diagram of a paramagnetic center further split by the influence of a nuclear spin. (A) represents the case where un > A 1/2 and (B) shows the splitting for n < A 1/2. The vertical axis is energy and the horizontal axis is magnetic field. 119

(4) 1/2 e + 1/2 un + 1/4 A

+ A/2

1/2 e - 1/2 un - 1/4 A

-1/2 oe + 1/2 un - 1/4 A - A/2 -1/2 Be- 1/2 iin + 1/4 A

(4 ) 1D - - I . IA A . 1 1/·, e ./A -.+ I/Z n

12+ n

1/2 'e - 1/4 A - 1/2 'n

-1/2 ue + 1/4 A - 1/2 on 2 - n -1/2 e -1/4 A + 1/2 n

m I = - 1/2 120

Figure 3. ENDOR spectra for (A) n > A 1/2 and (B) On < A /2. 121

A. IAI

1n

MHz

B. 2u n

IA 1/2

MHz 122

Figure 4. Rapid-passage dispersion-detected 35 GHz EPR spectrum of Hmv is shown in the upper trace, and the calculated derivative is given in the lower trace. Data collection parameters were 35.295 GHz microwave frequency, 2 K, 100 kHz modulation amplitude, 12 dB power, 64 ms time constant, 50 gain, 4 min sweep time. The specrum was simulated (dotted line) with gl,g2,g3 =

1.935, 1.865, 1.750. 123

...

......

______I I 13000 14000 15000

Mognetic Field [G] 124

Figure 5. The EPR spectrum of Hmv in the presence of DMSO is shown in the upper trace, and the calculated derivative is given in the lower trace. The simulated spectrum (dotted line) was generated from 25 % species (1 gl,g2,g3 =

1.95, 1.87, 1.75) and 75 %/ species 2 (gl,g2,g3 = 1.94, 1.87, 1.81). 125

1.86

1

1.94

12500 136000 13500 14000 14500

Madnetic FlVd Q] 126

Figure 6. 1H ENDOR spectra of Hmv at gl (1.94), g2 (1.87), and g3 (1.74). The three classes of protons are denoted by the dotted arrow (weakly coupled), the double-headed arrow (intermediately coupled), and the single arrows (strongly coupled). 127

1 1.94

'I,

1, I, 0

1.87 0

4. 1.74

______-20 -10 0 10 20

Frequency (MHz) 128

Figure 7. 1H ENDOR spectra of Hmv at gl = 1.94. Trace B is from a sample that was exchanged into D20. Experimental conditions were altered from conditions described for Figure 5 to obtain better resolution of the weakly coupled protons. Experimental parameters: 35.1 GHz microwave frequency; 2

K; 12 dB power; average of 470 (A) and 176 (B) scans. 129

A HO 2

B DO 2

-10 -6 -2 2 6 10

V-VH H 130

Figure 8. 1 H ENDOR spectrum of Hmv in the presence of DMSO at g2 = 1.87. The three classes of protons are denoted by the dotted arrow (weakly coupled), the double-headed arrow (intermediately coupled), and the single arrows (strongly coupled). 131

I I

I

_ · · · · 1 -20 -15 -10 -5 6 5 10 15 20 25 v - v(H)(MHz) 132

Figure 9. 14 N (A) and 15 N (B) ENDOR spectra of Hmv. N1 and N2 transitions are indicated by vertical lines. The u+ resonances are relatively intense (solid markings) and the u- transitions (dotted lines) are extremely weak and often not detected. The hyperfine-centered doublets of N1 are centered at A/2 as denoted by filled triangles and are separated by 2un. Larmor-centered doublets of N2 are centered at n (circles) and separated by A. Inverted triangles indicate 13C and 15N transitions at their respective Larmor frequencies due to the distant ENDOR effect. Conditions: 0.3 G modulation amplitude; 12 dB power; (A) 35.02 GHz average of 320 scans; (B) 35.09 GHz, average of 190 scans. 133

V

rN

T - l 1

-i -s m NI

A N2 _, _

15 N

V I

-- 2 4 6 e 10 12 14 16 I'a 2'0 2:2 Froeqwncy [MHz] 134

Figure 10. 14 N (A) and 15 N (B) ENDOR spectra of Hmv in the presence of DMSO. N1 and N2 transitions are indicated by vertical lines. The v+ resonances are more intense (solid markings) and the - transitions (dotted lines) are extremely weak. The hyperfine-centered doublets of N1 are centered at A/2 as denoted by filled triangles and are separated by 2n. Larmor-centered doublets of N2 are centered at Un (circles) and separated by A. Inverted triangles indicate 13C and 15N transitions at their respective Larmor frequencies due to the distant ENDOR effect. Conditions: 0.3 G modulation amplitude; 12 dB power; (A) 35.10 GHz average of 260 scans; (B) 35.90 GHz, average of 50 scans. 135

V,

A. 1 4 N

N2......

N1 ,! A14 KA..--

t NiaL

i I .- I1 -I t i , , , I

v v B. 15N

.2 4 a a lo0 12 *14 16 I 20 Frequency Hz) 136

Figure 11. 57Fe ENDOR spectra of the Fe(III) site at various g values for (A) Hmv and (B) Hmv and DMSO.23 The doublet is centered around A/2 and split by n (< 1 MHz). Simulations are shown by dotted lines. Spectrometer conditions: 35.2 GHz, 0.3 G modulation amplitude, average of 50-200 sweeps. 137

A B

...... 1.75 1.80

______.... 2. 1.81 1.85

...... - 1.87 1.90

?/ / . . . . . 1. 93 2 ...... 1.95 28 32 36 40 44 2r 3ue2 36 ( 40 Frequency (MHz) Frequency (Iz) 138

Figure 12. 57Fe ENDOR spectra of the Fe(II) site at various g values for (A) Hmv and (B) Hmv and DMSO as indicated by arrows. Spectra from unlabeled samples are denoted by dotted lines. Spectrometer conditions: 35.2 GHz, 0.3 G modulation amplitude, average of 50-200 sweeps. 139 57 Fe A 1

1.87

1.94

_ 8 1'2 16 20 24

Frequency (MHz)

B I

1.78

1.82

I- , 1.92

I

.95

I - 1 1 - 2 4 12 16 2'0 24 Frequency (MHz) 140

Figure 13. ENDOR spectra of Hmv at gl = 1.94 in the presence of (A) unlabeled

13 DMSO, (B) d6 DMSO, and (C) C DMSO. Larmor frequencies are as indicated, and the background resonances are due to 14N. Experimental conditions: 35.1

GHz (A) and (C), 35.0 GHz (B); 300 scans (A), 135 scans (B), and 200 scans (C). 141

A

(CHS)2SO

v (D)

B

(C*D3 )2 SO

v'v( (13C) C)

C

(*CH3)3 2 SO I I I I I I I I I i -r ! 6 7 8 9 10 11 12 13 14 15 16 17.18 19 Frequency MHz) 142

Figure 14. Mims pulsed ENDOR27 spectra of Hmv in the presence of isotopically labeled DMSO at g = 1.85. The spectra are centered at the respective Larmor frequences of 13C and 2H. Arrows indicate 1.2 MHz as the maximum hyperfine coupling for 13C and 0.8 MHz for 2 H. Conditions: 9.434 GHz, = 400 ns, ic/2 tp = 16 ns, tp(rf) = 60 s, sequence repetition rate 50 ms, average of 760 transients (13C) and 500 transients (2H). 143

1 C

-1.4 -1 -. 6 -. 2 .2 .6 1 1.4

Frequency (MHz)

y

2 H

-11 -16-I ' -.-22 ' ..2 ,.6 6 i I 1.4.4

Frequency (MHz) 144

Figure 15. 2H ESEEM spectra of Hmv in the presence of DMSO at g = 1.85. The time domain data and simulations are given in (A) through (C). The Fourier transformed frequency domain (D) spectrum and simulations are listed in (D) through (F). Conditions: 9.434 GHz, 3-pulse ESEEM sequence with z = 192 ns,

7r/2 tp = 16 ns, time T between first and second pulses stepped in 32 ns increments (256 total points), 50 ms repetition rate, 140 transients averaged. More details of these data can be found elsewhere.23 145

A C-l------5 ------uL-- Exerment

B v -- - -A Simulation - 3 2H

C Simulation - 6 2H ---L- __

I I · - . 20'00 6000 10000 T + (ns)

D

Experiment

E

Simulation - 3 2H F

Simulation - 6 2H

;L r 1 2 5 10 Frequency (MHz) 146

CHAPTER 4.

Radical Clock Substrate Probes and Kinetic Isotope Effect

Studies of the Hydroxylation of Hydrocarbons by Methane Monooxygenase 147 Introduction Methane monooxygenase (MMO) is a protein system employed by methanotrophic bacteria to oxidize methane to methanol, a process that provides these organisms with their sole source of carbon and energy (eq. 1).1 The soluble

+ + CH4 + 02 + NADH + H - CH30H + H2 0 + NAD (1)

MMOs from Methylococcus capsulatus (Bath)2 and Methylosinus trichosporium OB3b,3 two of the better studied systems, comprise three proteins. Included are a hydroxylase enzyme that binds substrate and dioxygen, a reductase that contains Fe2S2 and FAD moieties and accepts electrons from NADH, and a coupling protein that regulates electron transfer between the reductase and hydroxylase 4 and also modulates the substrate specificity5 6 and redox potentials 7 of the latter. Recently, much attention has been focused on the hydroxylase enzyme which, in addition to methane, is capable of hydroxylating a wide variety of substrates.8' 9 X-ray crystallographic studies10 of the diferric hydroxylase and ENDOR spectroscopic results1l for the the mixed-valent state reavealed that the active site contains a non-heme dinuclear iron center in which the metals are each coordinated to a histidine residue. One iron atom has two terminal monodentate glutamate ligands, and the other has one terminal monodentate glutamate and a water molecule in its coordination sphere. In the x-ray structure, the iron atoms are bridged by a hydroxide, a bidentate glutamate, and an exogenous acetate ligand. The catalytic mechanism of the MMO hydroxylase has frequently been compared to that of its widely studied heme analogue, cytochrome P-450.8'12-14 In P-450 catalyzed hydroxylations, it is postulated that a high valent iron-oxo, or ferryl, moiety is generated which abstracts a hydrogen atom from the substrate to 148 form a hydrocarbon radical that recombines with the coordinated hydroxyl radical before dissociating from the active site.15- 18 Especially important in establishing the presence of radical intermediates in this mechanism were studies with substrate probes that, following hydrogen atom abstraction to form a radical, isomerize to form, ultimately, a rearranged alcohol product.19-21 This process is illustrated for a specific probe, trans-2-phenylmethylcyclopropane (1), in Figure 1. Attempts to employ this methodology in studies of the hydroxylation of 1,1-dimethylcyclopropane with the MMO hydroxylase from M. trichosporium OB3b were consistent with radical formation, although cationic intermediates were also invoked.1 2 Other experimental evidence for the involvement of radical intermediates in the hydroxylation chemistry of MMO from the same organism has been reported. '8 13 22 In the present article we describe our investigations of the MMO hydroxy- lase from both organisms in which five substrate probes, 1- 5 illustrated in Figure 2, have been used to address the possibility of radical intermediates and other aspects of the hydroxylation mechanism. Specifically, reactions with trans- 1,2-dimethylcyclopropane (3) and bicyclo[2.1.0]pentane (4), both of which were used to study the rate constants for rebound with cytochrome P-450,19 20 and *withthree additional radical clock substrates, trans-2-phenylmethylcyclopropane (1) and 2,2-diphenylmethycyclopropane (2), and methylcubane (5), were carried out to determine whether substrate radicals are produced during hydroxylation. If, for example, hydrogen atom abstraction from probe 1 at the methyl position were to occur, the resulting cyclopropylcarbinyl radical (1U., Figure 1) would either be trapped before ring opening to give trans-(2- phenylcyclopropyl)methanol (la) or would ring open to the 1-phenylbut-3-enyl radical (1R). Subsequent trapping of radical 1R would afford 1-phenylbut-3-en- 1-ol (lb). Substrate 1 has been employed previously as a hypersensitive radical 149 probe in oxidations by resting cells and crude enzyme preparations of Pseudomonas oleovorans monooxygenase. 2 3 In that work, the only oxidation product detected was the ring-opened alcohol, leading to the conclusion that radical intermediates had been formed and that the oxygen rebound step had a rate constant of < 4 x 109 s-1 .

Experimental Section Isolation and Purification of Proteins. M. capsulatus(Bath) was grown as reported previously.24 25 The hydroxylase protein was purified and assayed fol- lowing the described procedure,2 5 except that Q Sepharose was substituted for DEAE Sepharose in the initial ion exchange chromatography step. Coupling protein B was prepared from an Escherichia coli strain harboring a plasmid containing this gene from M. capsulatus (Bath),26 and then purified according to published procedures. 27 The reductase was purified as described27 except that an HPLC TSKG3000SWG column was employed in the final purification step. Specific activities with propylene were measured at 200 to 250, 8000 to 8500, and 4000 to 4200 mU/mg for the hydroxylase, coupling protein, and reductase, respectively. Growth of M. trichosporiumOB3b and purification of its hydroxylase were carried out as reported. 2 8 Its specific activity for conversion of propylene to propylene oxide was monitored25 with the use of coupling protein and reductase from M. capsulatus (Bath), for which cross-reactivity with the M. trichosporium C)B3bhydroxylase has been previously reported.29 30 The measured activity was 180 mU/mg of protein, a value considerably less than expected,28 possibly due to the use of coupling protein and reductase from a different organism. Reactions of 15 iiM solutions of the M. trichosporiumOB3b hydroxylase with 10 mM hydro- gen peroxide produced 0.19 mM propylene oxide in a 3 min reaction, however, which compares favorably with literature reports that 44 gM hydroxylase and 10 150 mM H202 yielded approximately 0.4 mM propylene oxide in the same time pe- riod.14 The measured iron content, 2.2 mol Fe/mol hydroxylase, was less than found by others, 4.3 mol/mol protein,2 8 for which we have no obvious explana- tion. Synthesis and Characterization of Substrates and Products. The sub- strate trans-2-phenylmethylcyclopropane (1) was prepared as previously re- ported3 1 by reducing commercially available trans-2-phenylcyclopropanecar- boxylic acid or its ethyl ester to alcohol la, trans-(2-phenylcyclopropyl)methanol, with LiAlH4, converting the alcohol to the mesylate, and reducing the mesylate with LiEt3BH. As an alternative, conversion of alcohol la to the corresponding 3 2 chloride by treatment with Ph3P and CC14followed by reduction with LiAlH4 gave hydrocarbon 1 and ring-opened product 4-phenylbut-l-ene; treatment of the mixture with m-chloroperoxybenzoic acid to epoxidize the alkene by- product, followed by silica gel chromatography, gave pure 1. Deuterated analogues of 1 were prepared by the mesylate route with LiAlD4 as the source of deuterium. Their NMR and mass spectra were consistent with the expected amount of deuterium incorporation for each analogue. The substrate 2,2-diphenylmethylcyclopropane (2) was prepared by a se- quence similar to that described above for 1. Reaction3 3 of ethyl diazoacetate in the presence of CuSO4 with 1,1-diphenylethene gave ethyl 2,2-diphenylcyclo- propanecarboxylate that was reduced with LiAlH4 to (2,2-diphenylcyclo- propyl)methanol (2a). Conversion of 2a to the mesylate followed by LiEt3BH reduction gave 2. Methylcubane (5) was prepared as reported, along with its mono-, di-, and trideuterated analogs.34 Authentic samples of the products obtained by the MMO-catalyzed oxi- dation of substrates 1, 2, and 5 were prepared in the following manner. trans-(2- I'henylcyclopropyl)methanol la and its dideuterated analogue were obtained 151 from the synthesis of 1, and alcohol 2a was available in the preparation of 2, as indicated above. Monodeuterated la was prepared by LiA1D4 reduction of trans- 2-(phenylcyclopropyl)methanal which, in turn, was obtained from the acid by se- 35 quential treatment with H3BSMe2 and pyridinium chlorochromate. 1- Phenylbut-3-en-l-ol (lb) and 1,1-diphenylbut-3-en-l-ol (2b) were prepared by reactions of allylmagnesium chloride with benzaldehyde and benzophenone, re- spectively. Cubylmethanol (5a), along with acetylated products of reactions with 5, cubylmethanol acetate (5b), 2-methylcubanol acetate (5c) and 4-methylcubanol acetate (5d) were prepared from cubanecarboxylic acid.34 Dideuterated 5a was prepared as described elsewhere.34 An authentic sample of trans-2-(p-hydroxyphenyl)methylcyclopropane (lc) was prepared by the following sequence. Reaction33 of p-vinylanisole with ethyl diazoacetate gave a mixture of ethyl cis- and trans-2-(p- methoxyphenyl)cyclopropane carboxylates which was separated by column chromatography and crystallization. The trans isomer, identified by NMR spec- troscopy, was reduced with LiAlH4, and the resulting alcohol was converted to its mesylate which was then reduced with LiEt3BH. The resulting trans-2-(p- methoxyphenyl)methylcyclopropane was demethylated36 with NaSEt in DMF to give the desired product which was purified by preparative GC on a Carbowax 20M column. Authentic samples of the possible aromatic hydroxylated products from 2 were not prepared; no components consistent with these products were observed by G(Cmass spectral analysis of the oxidation mixtures. trans-1,2-Dimethylcyclopropane (3), trans-2-methylcyclopropylmethanol (3a), 4-penten-2-ol (3b), and 2-methylbut-3-en-l-ol (3c) were obtained from Wiley Organics (Coschocton, Ohio). Bicyclo[2.1.0]pentane (4) was synthesized according to a literature method. 3 7 152

Enzymatic Reactions. Reactions with MMO from M. capsulatus (Bath) were carried out at 45 °C with the reconstituted enzyme system in the following manner. Solutions were prepared containing 1.2 mg (10 gtM) of hydroxylase, 0.2 mg (24 gM) of coupling protein, and 0.4 mg (20 gM) of reductase, all in 25 mM MOPS (MOPS = 3-[N-morpholino]propanesulfonic acid), pH 7.0, in a final volume of 0.5 mL. A 0.5 gL (0.5 mg for 5) portion of substrate was then added by means of a microsyringe to achieve a maximum concentration of - 8 mM, most of the substrates not being completely soluble prior to conversion to products. The reaction mixture was placed in a 10 mL glass vial and sealed with a Suba-Seal septum. After addition of a 25 jiL portion of a 0.1 M stock solution of ethanol- free NADH to initiate the hydroxylation reaction, the vial was placed in a shaker- incubator maintained at 45 °C. Control reactions were performed in an identical manner except that no NADH was added. Reactions were allowed to proceed for 15 min, a time sufficient to afford at least 30 mol product per mol enzyme in those cases where authentic standards were available. Studies with the hydroxylase from M. trichosporiumOB3b and substrate 1 were carried out at 30 °C in one of two ways. Coupling protein and reductase from M. capsulatus(Bath) were incubated with the hydroxylase in the presence of NADH. The protein and NADH concentrations were the same as described in the preceding paragraph. Alternatively, reactions were performed by using 10 mM hydrogen peroxide and M. trichosporiumOB3b hydroxylase only, a known shunt for this enzyme.14 Substrate was added to the reaction mixture, which was then incubated as described above. Again, at least 30 turnovers were completed before the reaction was terminated. Instrumentation. Products from enzyme oxidations were analyzed with a Hewlett Packard (HP) model 5890 gas chromatograph equipped either with an FID detector or interfaced to an HP model 5971A mass spectrometer (EI, 70 eV). 153

For GC and GC /MS analysis of reactions with 1- 5, either a cross-linked FFAP (HP) or a cross-linked methyl silicon (HP) capillary column was employed, depending on which column gave better resolution of the reaction components. The column dimensions were 10 m x 0.5 mm x 1.0 jgm for GC analysis and 25 m x 0.2 mm x 0.33 pm for GC/MS analysis. Slightly different instrumental conditions were used to analyze the different reactions, as indicated in Table 1. Product Identification and Quantitation. Products were isolated by thorough extraction of the reaction mixture with diethyl ether or ethyl acetate. The septum of the reaction vial was removed and immediately replaced after addition of 5 mL of extracting solvent. The mixture was incubated for at least 10 min, the organic layer was then removed, and the entire procedure was repeated twice. The organic layers were combined, dried over anhydrous K2CO3, and then concentrated by slow evaporation under Ar to a volume of -100 gL. After the addition of an appropriate internal standard (vide infra), a 0.2 to 0.5 jILsam- ple of concentrated extract was injected into the gas chromatograph. The reten- tion times and mass spectra of the products were compared to those of authentic standards. GC retention times for reactants and products are listed in Table 2. Since 5a and methylcubanols are reportedly unstable, products formed in reactions of 5 were acetylated in the following manner to prevent decomposition. A small amount of pyridine and acetic anhydride (2 - 3 drops each) was combined with the concentrated extracts, and the resulting solution ( 0.5 ml) was stirred for 30 min. After the addition of a 1 ml portion of H20 the mixture was extracted three times with 2 ml volumes of methylene chloride. The CH2C12 extracts were combined, dried over K2CO3, concentrated, and analyzed as described above. Components of the hydroxylation reaction mixture for 1-3 and 5 were quantitated by constructing standard curves in the following manner. Solutions 154 containing known concentrations of a given component and a constant amount of an internal standard dissolved in methylene chloride or diethyl ether were injected into the GC. The ratio of the peak area corresponding to the component with that of the standard was then plotted against the concentration of added component in the sample. A linear plot was obtained and subsequently used as a calibration curve to determine the concentration of the component in extracts obtained from reactions with the enzyme. Plots were constructed in this manner for the alcohols la-3a and 5a, the rearranged alcohols lb-3b and 3c, 2-(p-hydroxy- phenyl)methylcyclopropane c, and for the substrates 1-3. Since no lb was detected in reactions of MMO from M. capsulatus(Bath) with 1 (vide infra), mass balance determinations were made to ensure that the amount of unreacted sub- strate and products corresponded to the amount of substrate added to the reac- tion. In addition, reactions containing 0.8 mM of lb as well as the usual quantity (8 mM) of 1 were carried out to determine if the latter would survive the hydroxyation conditions and/or compete with 1 for the enzyme. Control experiments were carried out without addition of NADH, and the substrate was recovered following the usual extraction and concentration procedures. Isotope Effect Studies. Reactions with MMO from M. capsulatus (Bath) were investigated with 1 and 5 and with their mono-, di-, and trideuterated derivatives at the methyl position. A V/K intramolecular isotope effect, measured under conditions in which substrate was not saturating, was investi- gated through studies of the mono- and dideuterated derivatives. The product alcohols la and 5a from reactions with each substrate were analyzed by GC/MS. For 1 the isotope effect was then calculated by comparison of the intensities, after statistical corrections, at m/z 148 and 149 for alcohols arising from the monodeuterated substrate and at m/z 149 and 150 for alcohols derived from the dideuterated substrate. For 5, the intensities at m/z (M - 19) 116 and 115 from the 155

monodeuterated substrate and at m/z (M - 19) 117 and 116 for alcohols from the dideuterated substrate were analyzed. V/K intermolecular isotope effects were evaluated by comparison of the rate of formation of la and 5a in MMO catalyzed reactions with the undeuterated and the trideuterated derivatives. For 1, the experiment was performed in two ways, as a competitive reaction and as separate reactions with each substrate. Competition experiments were performed with equal amounts (0.5 gl) of both derivatives present in the reaction vial. The mass spectral intensity at m/z 148 (la) representing reaction of the undeuterated substrate, was divided by the intensity at m/z 150 representing reaction with the trideuterated substrate, and the ratio was taken as the intermolecular isotope effect. In separate reactions with the two derivatives of 1, 2-(p-hydroxyphenyl)methylcyclopropane (c), a product of the enzymatic reaction (vide infra), was used as an internal standard for the purpose of quantitation. The amount of la relative to lc was calculated for each derivative. The ratio of alcohol to phenol for the undeuteurated derivative was divided by the same ratio for the trideuterated derivative to provide the intermolecular isotope effect. Only separate reactions were performed with substrate 5, and the internal standard trans-2- methylcyclopropanol was used to quantitate the amount of product formed. Results Table 3 reports the results of the catalytic hydroxylation of substrates 1 - 5 by MMO from M. capsulatus (Bath). For substrates 1 - 5, the ratio of experimen- tally observed alcohols, corresponding to hydroxylation without and with rear- rangement, is indicated. In addition, the table includes the known rearrange- ment rates of the appropriate radical intermediates. Chromatographic retention times and typical GC traces for components of the reaction mixtures are provided in Table 2 and Figure 3, respectively. 156

Reactions with 1. The hydroxylation of 1 yielded two products as revealed by both GC and GC/MS analysis. They were identified as the alcohol la and 2-(p-hydroxyphenyl)methylcyclopropane (lc)38 by comparison of their retention times and mass spectra with those of authentic standards. Quantitation of the peaks revealed that the two products are formed in approximately equal concentrations and at equal rates over a 20 min reaction period, as illustrated in Figure 4. To ensure that all added material was accounted for in the analysis, mass balance studies were carried out. The amount of material recovered in control reactions run in the absence of reductant corresponded to 84.4 + 0.6% of the substrate initially added, the remainder apparently being lost through the various extraction manipulations. When NADH was present to initiate and sustain enzyme turnover, 85.0 ± 0.5% of the material added was recovered, 5.0 ± 0.2% of which was la and 5.1 + 0.1%, c. No rearranged alcohol lb was detected. The possibility that lb formed but was unstable under the hydroxylation conditions was investigated through analysis of a reaction containing 8 mM of 1 and 0.8 mM of lb. In the presence of NADH, 78.6 + 1.3% of lb was recovered, compared to 78.2 + 1.0% in control reactions without NADH. This result indicates that lb is unaffected during the hydroxylation reaction. The yields of la and lc remained unchanged. Hydroxylation studies of the mono- and dideuterated derivatives of sub- strate 1 gave values for kH/kD of 5.15 ± 0.38 and 5.03 + 0.42, respectively, for the intramolecular isotope effect. The intermolecular isotope effect was determined by comparing results for 1 with those for its trideuterated derivative. A value of 1.18 + 0.15 was obtained for reactions that were performed separately whereas competitive reactions performed with both derivatives present in the reaction mixture yielded a value of 0.89 0.10 (see Experimental Section). In the competition study, the ratio of phenols (lc-do:lc-d 3) was 1.1. 157

Reactions with 2. GC analysis of hydroxylation reactions with 2 revealed only a single product which, by comparison to an authentic standard, was identified as the alcohol 2a. No peaks that might arise from rearranged alcohol 2b or aromatic hydroxylation products were detected. A plot showing the rate of product formation is given in Figure 5. Reactions with 3. Analysis of the extracts from reactions with trans-1,2- dimethylcyclopropane (3) revealed only one product, the concentration of which increased linearly with time over a 10 min period, as plotted in Figure 4. It was identified as the unrearranged alcohol, trans-2-methylcyclopropylmethanol (3a), through comparison of its GC retention time with that of an authentic standard. No 4-penten-2-ol (3b) or 2-methylbut-3-en-l-ol (3c) was detected. Reactions with 4. The substrate bicyclo[2.1.0]pentane (4) afforded 3 products upon reaction with MMO (Figure 6), each of which had its parent ion at nz/z 84 as determined by GC/MS analysis. No authentic standards were available for the expected products, but one product could be positively identified as endo-2-hydroxybicyclo[2.1.0]pentane (4a) by comparison of its mass spectrum with one published previously.19 The second major peak in the gas chromatogram was assigned as exo-2-hydroxybicyclo[2.1.0]pentane (4b). Unfortunately no mass spectral data for this compound were available for comparison. The peak had approximately the same intensity (-85%) in the total ion chromatogram of the reaction extracts as that of 4a. Its mass spectrum differed from those of 4a and 3-cyclopenten-l-ol (4c), for which mass spectral data are also available.39 Hydroxylation of 4 by cytochrome P-450 yielded only 4a and 4c.19'20 The failure of this enzyme to afford 4b was attributed to constraints at the active site. It may be that the active site of MMO is less dis- criminating, which could account for the formation of both 4a and 4b. 158

The third product from reactions with 4 was present in only trace amounts and was assigned as 3-cyclopenten-l-ol (4c). It has been previously reported that 4a undergoes thermal rearrangement to 4c.19' 20 In particular, when 4a isolated from the hydroxylation of 4 by MMO was injected into the GC/MS, the ratio of 4c to 4a was found to be ~1:30. Compound 4b is expected to behave similarly. The intensity of the peak corresponding to 4c was also found to increase relative to those for 4a and 4b when the reactions were run at 45 °C for longer times (Fig. S3). Increasing the injector and oven temperatures of the gas chromatograph produced a similar effect. The ratio of 4c to 4a was 1:40, or, including both 4a and 4b, 1:80, and increased with longer incubation times at 45 °C to about 1:15. It is therefore likely that a significant amount, if not all, of 4c obtained in these ex- periments is due to thermal rearrangement of 4a and 4b. The amount of 4c pre- sent in the enzymatic reactions is small enough that its presence could be solely the result of thermal rearrangement of 4a and 4b.19 Reactions with 5. In reaction extracts of 5, GC/MS analysis revealed the presence of only one product, 5a, which was stable for up to a week under the extraction and GC/MS conditions employed in this work. GC/MS analysis of the acetylated extracts contained only unrearranged 5b. No products indicative of radical rearrangement (5e) or hydroxylation at another position (5c or 5d) were observed, either in the extracts or following acetylation. The mono- and dideuterated derivatives of 5 gave kH/kD ratios of 5.29 + 0.32 and 4.21 ± 0.46, respectively, revealing a substantial intramolecular isotope effect. Separate reactions performed with the undeuterated and trideuterated derivatives present resulted in a value of 0.87 + 0.15. The magnitudes of both the intermolecular and :intramolecuar isotope effects for 5 are in good agreement with those obtained for

1. 159

Experiments were also carried out with 1 and the hydroxylase from M. tri- chosporium OB3b. In contrast to reactions with MMO from M. capsulatus (Bath), three products were observed. They were identified as the unrearranged alcohol la, 2-(p-hydroxyphenyl)methylcyclopropane (c), and the rearranged product lb (Figure 3). The ratio of unrearranged to rearranged alcohol was 32:1 in reactions performed with coupling protein and reductase from M. capsulatus (Bath) and 22:1 for reactions using hydrogen peroxide. Discussion Analysis of Products Formed. In this work, five hydrocarbon substrates were employed as mechanistic probes in oxidations catalyzed by the MMO of M. capsulatus (Bath). In each case, if a hydrogen atom were abstracted from the probe, the resulting radical could either rebound to form the unrearranged prod- uct or ring open to afford the rearranged product (Figure 1). In no case was re- arrangement observed. For trans-1,2-dimethylcyclopropane (3) only a single product formed, corresponding to the unrearranged alcohol (3a, eq. 2). No allylic alcohol was detected (3b, eq. 3), implying that no more than 1% of this product Phi-HA t trapping

CH3 CH3 CH3 CH CH 33 CH20H (2) 3 U 3a

rearrange- . trapping OH ',,ACH ment CH 3 CH nt CH 3 CH 3 ) 3b formed during the hydroxylation reaction. From the known rate constant for :rearrangement of the corresponding cyclopropylcarbinyl radical (Table 3),21and assuming that this rate constant is the same for substrate free in solution and bound to the enzyme (vide infra), we estimate that the rebound rate constant of a 160 radical intermediate would have to be at least 4 x 1010 s-1 at 45 °C. A similar analysis21'40-42 of the results for reactions with compounds 4, 5, 1, and 2, raises the rebound rate constant limits to 3 x 1011, 8 x 1012, 4 x 1013, and 1 x 1013 s- 1, respectively. The latter two values exceed the rate constant of decomposition of a transition state and are incompatible with a discrete radical intermediate. The rate constants for ring opening of radicals derived from 1 and 2 (eq. 4) have been measured indirectly by competition against PhSeH trapping.41 '4 2 At room temperature, the ring openings have rate constants of 3 x 1011 s-1 and

Ph ~trapping-H. P

R CH, R 'CHCH 2 ' R 2OH (4a) R=H, 1 R = H, la R = Ph, 2 R = Ph, 2a

OH Ph. ,A rearrange- Ph ... trapping ph ., , (4

R J"CH, ment R ' R = H, lb R = Ph, 2b

5 x 1011s -1, respectively, which places these reactions among the fastest reported radical rearrangements involving a bond breaking step. With such a velocity, detectable amounts of ring-opened products are expected to be produced from these radicals even when the fastest possible trapping reactions compete. Therefore, the absence of ring-opened alcohols lb and 2b suggests that probes 1 and 2 were converted directly to alcohols la and 2a without formation of radicals. On the other hand, a small amount of rearranged product lb was ob- served in the studies of MMO from M. trichosporiumOB3b. From the ratio of un- rearranged to rearranged alcohol the rate constant for rebound was calculated to be 6-10 x 1012s -1. The observed differences in the results for MMO from the two different sources will be discussed later. 161 In the case of the chiral substrate probe 1, oxidation occurred not only at the methyl group but also at the phenyl ring to form 2-(p-hydroxyphenyl)- methylcyclopropane (c) (Table 3).38 The fact that la and lc are produced in equal quantities over the course of the reaction suggested that the two products might arise from different enantiomers of the probe. Studies with partially resolved (-90 "%o)1 indicate that, with R,R 1, twice as much of la is produced than lc. With S,S 1, however, c is present at twice the levels of la. The analysis of these results is complicated by the possibility that formation of la and lc may proceed with different rate constants. In addition, a particular rate constant may vary with each enantiomer of the probe. It is therefore difficult in the absence of this quantitative information to draw firm conclusions from this experiment. A system in which only one product is produced would be better for studying the chiral discrimination of the site. Nevertheless, it would appear at least qualitatively that the enzyme can discriminate between the two enantiomers to achieve regioselective, if not regiospecific, hydroxylation. Deuterium Isotope Effects. Studies of the reaction of deuterated deriva- tives of 1 and 5 with MMO from M. capsulatus (Bath) failed to reveal any intermolecular isotope effect. Both individual and competitive reactions with the undeuterated and trideuterated derivatives of 1 yielded values of (kH/kD)obs of approximately 1.0 and in good agreement with one another, considering the error limits. Similarly, the ratio in the separate experiments with undeuterated and trideuterated 5 was 0.87. For 1, the ratio of undeuterated and trideuterated phenols observed in the competition experiment was 1.1. When combined with the ratio of 0.89 for the undeuterated and dideuterated alcohols la, this result shows that the overall rates of oxidation of the undeuterated and trideuterated substrates were equal. Thus, there was essentially no kinetic discrimination for substrates by the enzyme, and this behavior suggests that C-H bond breaking is 162 not rate-limiting in the overall hydroxylation reaction. Such a result is consistent with some,13'4 3 but not all,44 previous studies of MMO hydroxylation reactions using deuterated substrates.

Given that the overall oxidation rates of 1-do and 1-d3 were the same, the product distributions should be considered in more detail. If oxidation of each of the enantiomers of substrate 1 could have occurred at either the methyl group or aromatic ring, and if rotation can occur in the active site, then a kinetic isotope effect should have been observed in oxidations of the undeuterated versus trideuterated probes with substantially more phenol product being formed in the latter case. This result is predicted from the intramolecular kinetic isotope effects that were found (vide infra). That such was not the case reinforces speculation that products la and c arise from different enantiomers of the probe. This effect could be masked, however, if reoriention of the substrate radical prior to hydroxylation is hindered, since the two possible positions for hydroxylation are very far apart and hydroxylation at the other site would most likely involve substrate movement. In contrast to the lack of an intermolecular isotope effect, a significant in- tramolecular isotope effect of 4.8 to 5.1 (statistically corrected) was observed in oxidations of both the mono- and dideuterated probes 1 and 5. In each case, the observed isotope effects are equal to the primary kinetic isotope effects (kH/kD) divided by a secondary kinetic isotope effect that results from the fact that an additional deuterium atom remains bound after oxidation of a C-H bond. The data do not permit an extraction of the secondary kinetic isotope effect, but it is clear that the (kH/kD)obs ratio signals a substantial C-H bond stretching component in the transition state of the rate-limiting step in the oxidation reaction. The value of (kH/kD)obs= 5.1 for 1 and 4.8 for 5 may be compared with the intramolecular isotope effect of 7.8 observed in the oxidation of probe 1 with 163 P. oleovorans monooxygenase. 2 3 Recently, a value for kH/kD = 4.2 ± 0.2 was reported for MMO from M. trichosporiumOB3b with the substrates (S) and (R)-[1- 2H,1-3 H]ethane, in good agreement with the present results.45 Cytochrome P- 450 dependent enzymes oxidize a number of substrates with intramolecular isotope effects of 7-14.20 Intramolecular isotope effects for hydrogen atom abstractions by the reactive tert-butoxy radical studied at temperatures in the vicinity of 25 C range from 1.2 to 5.4, and isotope effects for hydrogen abstractions by less reactive peroxy radicals are greater.46 Mechanistic Considerations

Formation of the Dioxygen-Activated Diiron Center. Figure 7 outlines a reasonable working hypothesis for the catalytic mechanism of MMO. Substrate first binds to the complete system containing all three protein components. Addition of NADH then effects a two-electron reduction of the hydroxylase from the oxidized Fe(III)Fe(III) to the fully reduced Fe(II)Fe(II) form, bypassing the inactive4 7 Fe(II)Fe(III) state.7'44 The fully reduced hydroxylase next reacts with dioxygen in a two-electron step to form a diiron(III) peroxide complex which, either itself or, upon transformation via two more electron transfer steps at the diiron center, is sufficiently activated to attack the hydrocarbon substrate. The nature of this transformed species, if it exists, is currently unknown, but it could involve a high valent iron oxo moiety (Figure 8), by analogy with that proposed for cytochrome P-450.15 The second iron atom in the MMO hydroxylase active site would stabilize such a unit through redox charge delocalization, as shown in Figure 8, in much the same fashion that cytochrome P-450 is thought to facilitate the formation of a high valent iron oxo species through oxidation of the por- phyrin ring to a r-cation radical. Alternatively, the internal two-electron transfer step could produce an active site radical (R'.), such as the hydroxyl radical or a ligand-based radical involving an amino acid residue, and an iron(IV) center that 164 would again be stabilized by tautomerization and electron transfer involving the other iron atom (Figure 8). Although there is presently no direct evidence for such a radical species in the MMO hydroxylase active site, the combination of a redox active metal with a protein derived radical to effect oxygen-activated redox transformations is known in the related iron enzyme ribonucleotide reductase,48 in the heme peroxidase prostaglandin H synthase,4 9 and in the copper enzyme galactose oxidase.50 A third possibility is that the diiron(III) peroxide unit itself might be sufficiently activated to carry out the hydroxylation reaction. As depicted in Figure 8, the peroxide ligand might perhaps be coordinated to the diiron(III) unit in an r2,q 2 fashion, analogous to the binding mode thought to be important in the dioxygen transport dicopper enzyme hemocyanin. 51,52 Other modes of peroxide binding to one or both iron atoms are possible, as indicated in Figure 9. Several important model studies have provided spectroscopic evidence for the existence of diiron(III) peroxides;5 3-6 0 and, related species,5 3' 61' 62 including a high valent iron oxo intermediate, 6 3 have been implicated in hydroxylation and epoxidation reactions. None has yet been structurally characterized, however. Once the activated iron center, whatever it may be, is discharged, product is released with concomitant formation of the diiron(III) form of the hydroxylase that enters another cycle in the catalysis (Figure 7). A peroxide shunt, analogous to that found in the P-450 enzyme, has been reported for the M. trichosporium (OB3b) MMO14 and confirmed by us in the present work. As indicated in Figure 7, the use of hydrogen peroxide permits hydroxylation reactions to occur in the absence of the coupling and reductase proteins. Addition of hydrogen peroxide to hydroxylase isolated from the M. capsulatusorganism, however, displays only 5 to 10% of the activity measured with the complete, three protein system.64 165 The Hydroxylation Reaction. Figure 10 presents six possible mechanisms for substrate hydroxylation following generation of the dioxygen-activated diiron center. In all cases except the last, 0-0 bond cleavage occurs prior to the C-H bond-breaking step. The six possibilities are (A) direct insertion of the oxygen atom of a high valent iron oxo species into a C-H bond; (B) concerted addition of the C-H bond to the high valent iron oxo species to form a metal- carbon bond followed by reductive elimination of the alcohol; (C) heterolytic attack of a high valent iron oxo species on the R-H bond followed by recombination. to afford product; (D) homolytic attack of a high valent iron oxo species on the R-H bond followed by return of the hydroxyl group from iron to the alkyl radical, a so-called oxygen rebound step; (E) abstraction of a hydrogen atom from the substrate by hydroxyl or another radical within the active site to form an alkyl radical which then adds to the iron-bound oxygen atom; and (F) electrophilic attack of an rI2,n 2-peroxide or related species (Figure 9) on the substrate to form a carbon-oxygen bond followed by release of product. Mechanisms I) and E involve transient formation of a substrate-derived alkyl radical, which could rearrange as shown in Figure 1, and it is these pathways that are directly addressed by the present investigation. The carbocation shown in C (2) could also rearrange, and its involvement in the hydroxylation mechanism would similarly be revealed by our studies. Experiments reported here and previously 9' 13 with MMO from M. tri- chosporium OB3b indicate that a radical is formed in the catalytic cycle and, a paper has recently appeared reporting results that are consistent with a radical intermediate.4 5 No ring-opened products are observed during the hydroxylation of substrate probes 1 - 5 with MMO from M. capsulatus (Bath), however. There are several possible explanations for these results. The simplest is that radicals and carbocations do not form and that pathways C (2), D and E can be excluded 166 for the reaction mechanism for MMO from this organism. Alternatively, radicals could form but recombine rapidly with the high valent iron-oxo moiety before they have a chance to ring open. Although this alternative is reasonable for probes 3, 4 and possibly 5, it is not a viable explanation for 1 and 2 since it would require a radical rebound rate constant greater than 4 x 1013s -1. This conclusion is only valid, however, provided that the hydroxylase active site does not perturb the ring opening rate constant of probes 1 and 2. Moreover, there are other potential problems arising in probe substrate oxidations within enzyme active sites, as delineated in a recent report of cytochrome P-450 dependent enzyme ox- idations. 20 In that study, two "poorly behaved" probes gave much less than the expected amount of rearranged product. In addition to decreasing the rate constant for radical rearrangement owing to steric effects, the low yields of rearranged products might arise from a possible change in mechanism, for example the onset of an insertion reaction, an increase in the rate constant of oxygen rebound, or reaction of the rearranged radical with another site on the enzyme. Two of these potential problems can be excluded as the reason for the fail- ure to detect ring-opened alcohols in the present study. Because the rearrange- ment of radical 1U. to 1R. (Figure 1) is fast enough to compete with any other process, it is not possible that acceleration of the oxygen rebound step without a diminution in the rate constant for the radical rearrangement could subvert the rearrangement. Furthermore, if ring-opened radicals were to react with another position in the enzyme active site, then the probe would most likely have deacti- vated the enzyme as a suicide substrate inhibitor. Although an extensive analy- sis of enzyme activity was not conducted for these substrates, there did not ap- pear to be a noticeable loss of activity over the 15 min time course in these stud- ies. 167 The other possibility, that constraints of the enzyme active site alter the rate constant for radical rearrangement, can be considered for each substrate. For probe 5, abstraction of a hydrogen atom at the methyl position leads to cubylmethyl radical 5U, as shown in Figure 11. Rearrangement of this species yields 5R*. Inspection of 5R. reveals that the substrate radical has undergone rearrangement, yet the radical is left at the methyl position. No skeletal rearrangement of carbon atoms occurs during this rearrangement, nor is there any requirement for substrate reorientation for a subsequent rebound reaction.6 5 Steric constraints of the active site should be considered a negligible influence on the rate constant for radical rearrangement. For probes 1 and 2, it is possible that the orientation of the phenyl group in enzyme-bound probe 1 is controlled by the steric confinement of the active site such as to preclude co-alignment of the 7r-system of the phenyl ring with the bond of the cyclopropane ring that is cleaved. The imposition of such a stereoelectronic effect on the ring opening reaction might result in a marked reduction in the rate constant for ring opening of the radical derived from 1. In the extreme case where the it-system of the phenyl ring is oriented perpendicular to the cyclopropyl ring bond that is opened, the contribution of a phenyl resonance effect to the cyclopropylcarbinyl bond breaking step would be eliminated, leaving only an inductive effect. The manifestation of an active site constraint-enforced stereoelectronic ef- fect on the kinetics of ring opening can be estimated semi-quantitatively via Marcus theory..40° 66- 69 It has been shown42 that the rate constants for reactions of the parent system, ring opening of the cyclopropylcarbinyl radical (6) to the 3- butenyl radical (7) and cyclization of radical 7, can be used to predict rate constants for ring openings of substituted cyclopropylcarbinyl radicals. In brief, the rate constant for ring opening of 6 at 25 °C (kr = 5 x 107 s-1 for cleavage of one 168 bond) 70 -73 and that for cyclization of 7 (kr = 8000 s-1)7 4-7 6 give AG° = -5.2 kcal/mol; AGt has been measured to be 6.9 kcal/mol. Solving the Marcus equation (eq 5) with these values gives AGti = 9.32 kcal/mol, the intrinsic

AGt = AGti + AG°/2 + (AG) 2/16AGti (5) activation energy expected for a thermoneutral cyclopropylcarbinyl radical ring opening. This value of AGit can be used in eq 5 to calculate AG$ for cyclopropylcarbinyl ring openings of varying exergonicity. Figure 12 shows a plot of log kr as a function of the exergonicity of the reaction. Comparisons of standard bond dissociation energies (BDE)can be used to estimate the exergonicity of the cyclopropylcarbinyl ring-opening reactions. The BDE for a benzylic hydrogen in toluene is 10-11 kcal/mol smaller than that of a primary C-H bond in ethane or propane.77 78 For the benzylic position in ethyl- benzene, the BDE is reduced by approximately 2.5 kcal/mol from that of toluene. 77 78 Thus, the ring opening of the radical derived from 1 is expected to be about 12.5-13.5 kcal/mol more exergonic than the value of the radical of 6, giving a value of AG0 = -18.2 kcal/mol. If we assume that the full overlap of the r-system of the phenyl ring is available in the transition state for ring opening of the radical derived from 1, then the predicted rate constant for ring opening of this radical at 25 °C is 1 x 1011s -1. Given the crude nature of the analysis, this value compares favorably with the observed rate constant of 3 x 1011s -1. If the phenyl group in the radical derived from 1 is locked such that its n- system cannot overlap fully with the breaking bond in the cyclopropyl ring, the resonance stabilization would be a function of cos2 0, where 0 is the dihedral an- gle of the breaking bond and the tc-systemof the phenyl ring. When the latter is orthogonal to the breaking bond (0 = 90°), the effect of the phenyl group would 169 be reduced to approximately that of the inductive effect of an alkyl group. In this extreme case, the appropriate model reaction for the Marcus analysis would be one in which the exergonicity of the ring-opening reaction is only about 2.5-3 kcal/mol greater than the value for 6 based on the differences in BDE between 1° and 2 alkane C-H bonds.77 This value gives a predicted rate constant at 25 C of about 4 x 108 s-1 . Using the two limiting cases discussed above, corresponding to phenyl stabilization between 3 and 13 kcal/mol, the stereoelectronic effect of an enforced position of the phenyl group in the radical derived from 1 is shown on the plot in Figure 12 for selected dihedral angles. When 0 is 60°, the expected rate constant for ring opening at 25 C is reduced to 2 x 109 s, which is about an order of magnitude less than the oxygen rebound rate constants found in P-450 oxidations. 20 Because it is possible that an oxygen rebound rate constant in the MMO hydroxylase is faster than that found in the P-450 studies, further reduction of the rate constant for ring opening of radical derived from 1 could lead to undetectably small amounts of ring-opened alcohol product lb. Thus the absence of lb from oxidation of probe 1 could result from an extreme alteration in the rate constant for ring opening of the derived cyclopropylcarbinyl radical in the enzyme active site.79 80 Although the Marcus analysis demonstrates that a stereoelectronic reduc- tion in the rate constant for ring opening of a radical derived from 1 is a possible explanation for the absence of ring-opened alcohol lb, it should be recalled that no ring-opened product formed in the hydroxylation of the closely related sub- strate 2. A significant reduction in the rate constant for ring opening of the radical derived from this substrate probe due to steric effects in the active site is less likely. In this case, both phenyl groups must be held such that their - systems are virtually orthogonal to the breaking bond in order to reduce 170 resonance stabilization in the transition state for opening, a much less probable situation. Moreover, such an arrangement would require that the phenyl groups approach coplanarity with one another, a geometry that would increase steric interactions between them. The results from probe 2 therefore suggest that a radical or carbocation is not formed and that the hydroxylation mechanism involves one of the alternative pathways A, B, C (1), or F in Figure 10. Moreover, a recent measurement of the ring-opening kinetics of several constrained, aryl- substituted cyclopropylcarbinyl radicals revealed that severe dihedral angles do not appreciably diminish the rate constants for ring opening.8 1 This study strongly implies that non-bonding steric effects in the active site are unlikely to diminish the ring-opening rate constant. There is another factor to be considered with regard to the results with probe 1. The discussion of steric effects on the rate constant for ring opening of substrate derived radical ignored the fact that two enantiomers of this substrate were allowed to react with the chiral enzyme. If sterics-enforced stereoelectronic effects were operative, it is unlikely that both enantiomeric radicals derived from the enantiomers of the probe would suffer similar ring-opening rate retardations. The formation of aryl hydroxylated product in amounts comparable to the amount of alcohol la produced complicates the analysis, however. As indicated above, it is likely that the two different products are derived from the two differ- ent enantiomers of the probe substrate. Aside from steric factors within the active site, other considerations to ex- plain the lack of rearranged substrate with M. capsulatus (Bath) must be ad- dressed. For example, one cannot rule out that the hydroxylation reaction may proceed through two different mechanisms from a common intermediate. One pathway, D or E, could involve a substrate radical, thus accounting for the rear- ranged products observed with MMO from M. trichosporiumOB3b. An alterna- 171 tive pathway could involve A, B, C, or F. The specific pathway chosen would then depend on the energetics of the substrate. It is possible, however, that the frequency with which each pathway is followed may differ between the two organisms. Alternatively, MMO from M. capsulatus (Bath) and M. trichosporium OB3b may exhibit different hydroxylation mechanisms, which would explain the presence of ring-opened products for the latter. We now return to the intramolecular deuterium isotope effects of 5.1 and 4.8 observed for the hydroxylation of deuterated 1 and 5. Although an isotope effect of this magnitude is generally associated with processes that involve a relatively linear transition state,8 2 such as would occur for oxygen rebound, mechanisms containing intramolecular cyclic transition states cannot be excluded. In fact, dimethyldioxirane, a reagent believed to oxidize hydrocarbons 83 via an insertion process, oxidized cyclododecane-do and -d24 with an isotope effect of 5.84 Thus our observed value of 5.1 would be consistent with pathways A, B, C (1), or F, all of which involve transition states for C-H bond cleavage that resemble the dioxirane oxygen insertion process and avoid the formation of a substrate radical or carbocation in the catalytic cycle.8 2 The previous observation of an NIH shift in the hydroxylation of aromatic substrates with MMO from M. capsulatus(Bath) would support such a mechanism.8' 85 In summary, the failure of substrate probes 1, 2, and 5 to form rearranged alcohol products upon hydroxylation with the M. capsulatus(Bath) MMO is best interpreted in terms of a mechanism that does not involve the formation of a radical or carbocation, namely, A, B, C (1), or F. It is unlikely that the enzyme active site induces a stereoelectronic barrier to ring opening. The fact that the same amount (85%) of the probe, both converted and unconverted, can be recovered from the enzymatic reaction mixture under hydroxylation conditions or in control reactions omitting NADH indicates that ring-opened substrate 172 radical has not formed and been lost in the extraction procedure. In addition, these probes do not lead to inactivation of the enzyme over the time course of the reaction, indicating that ring-opened product has not formed and reacted with the enzyme. The kinetic isotope results are consistent with a concerted pathway for C-H bond insertion for the key hydroxylation step. Comparisons with Related Work. The MMO mechanism is almost al- ways drawn in analogy to that of cytochrome P-450.8' 13 14 In particular, a cat- alytic cycle involving an iron-oxo intermediate (Figure 8) that abstracts H. from the alkane (mechanism D, Figure 10) has been proposed by several authors. The ability of high concentrations of hydrogen peroxide to effect a shunt pathway for the M. trichosporium OB3b MMO has been cited as evidence for heterolytic cleavage of the 0-0 bond, 14although it is noteworthy that, unlike the situation for cytochrome P-450, other oxo transfer reagents could not be substituted for

H20 2 . Hydrogen peroxide does not afford comparable levels of product in the M. capsulatus (Bath) system.6 4' 86 Various authors have invoked radical intermediates for the hydroxylation reaction. For example, exo, exo, exo, exo-

2,3,5,6-d4-norbornane epimerizes upon hydroxylation by M. trichosporiumOB3b MMO, 13 a result that has its parallels in cytochrome P-450 chemistry.87 The extent of epimerization with MMO, however, was significantly less, being 2% following hydrogen atom abstraction from the endo position compared to 18% with cytochrome P-450, and 5% after abstraction at the exo position as compared to 14% with cytochrome P-450. Similarly, allylic rearrangements occurred in 20% of the MMO hydroxylation products from 3,3,6,6-d4-cyclohexene compared to 33% for cytochrome P-450.13'88 Mechanistic studies of MMO from M. trichosporium OB3b with the free radical probe 1,1-dimethylcyclopropane and with (S) and (R)-[1-2H,1-3 H]ethane were also consistent with radical formation, but cationic intermediates could not be ruled out.12'45 It has also been claimed 173 that radical intermediates can be trapped during methane hydroxylation by the MMO of M. capsulatus (Bath).22 The mechanism for olefin epoxidation by MMO from M. trichosporiumOB3b differs from that believed to occur with cytochrome 1P-450.13 With cytochrome P-450, the 1-trans-proton of propylene exchanges with solvent protons during turnover,89 leading to the hypothesis that the epoxidation mechanism involves oxametallocycles and iron carbene intermediates. In the reaction of propylene with MMO, however, no such exchange occurred.13 In view of these and the present results, it would appear that no single unifying mechanism can account for all of the data. Instead, it seems likely to us that more than one mechanism might be operative, possibly from a common branchpoint. The frequency with which a specific pathway is followed would depend on factors such as the steric demands of the substrate at the active site, the organism from which the MMO is isolated, and even the temperature at which the reaction is carried out. There are differences in the coupling proteins obtained from the M. capsulatus (Bath) and M. trichosporium OB3b organisms, 9 0 9 1 which could alter the properties of the system. Moreover, it should be recalled that the M. capsulatus(Bath) methanotroph was isolated from thermal waters and has its optimal monooxygenase activity at 45 C, whereas the MMO from M. tri- chosporium OB3b is typically assayed at ambient temperature (23 C). Further studies, involving additional mechanistic probes and the application of other methodologies, are currently in progress to provide further insight into these is- sues. Conclusions. The major findings of this investigation may be summa- rized as follows: (1) The absence of ring-opened products in the hydroxylation of probes 1, 2, and 5 suggests that the mechanism for MMO from M. capsulatus (Bath) may not involve radicals or carbocations, at least for these substrates. 174

(2) Isotope effect studies with deuterated probes 1 and 5 indicate that the hydroxylation step is not rate determining in the overall MMO mechanism but that substantial C-H bond activation is involved in this step. (3) The observation that equimolar quantities of phenol and alcohol are produced in the hydroxylations of racemic mixtures of 1 and its trideuterated analogue implies that chiral discrimination in the enzyme active site can dictate the regiospecificity of the reaction. (4) A semi-quantitative Marcus theory analysis was used to evaluate how the ring-opening rate constant of phenyl substituted cyclopropanes can be tuned by changing the dihedral angle between the phenyl and cyclopropyl rings. (5) Several detailed proposals have been set forth for two discrete arcs of the MMO catalytic cycle (Figure 7), the activation of the reduced, diiron(II) form by dioxygen (Figure 8), and the subsequent hydroxylation step (Figure 10). These considerations should serve as a valuable reference point for the interpre- tation of mechanistic results from diiron oxo and related monooxygenases as well as their model compounds. (6) Analysis of the present and literature results for the hydroxylation of hydrocarbons by MMO suggests that multiple mechanisms may be operative, possibly from a common branch point.

Acknowledgments. Substrate probes and authentic standards were provided by C. C. Johnson and Professor M. Newcomb at Wayne State University. Professor Newcomb also contributed the Marcus analysis of the ring- opening rate constants of the phenyl substituted cyclopropanes. Most of this 'work was published previously. Thanks also to Athanasios Salifoglou for providing cell paste. 175 References

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(34) Choi, S.-Y.; Eaton, P. E.; Hollenberg, P. F.; Liu, K. E.; Lippard, S. J.; Newcomb, M.; Putt, D. A.; Upadhyaya, S. P., manuscript in preparation.

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1969, 49, 1-6. (38) It is possible that the ortho and meta isomers of c coeluted with lc in the GC analysis and were thus not detected. Separation of enzymatic reaction mixtures with 1 by HPLC (C-18 reverse-phase) yielded only 3 peaks which corresponded to 1, la, and c. It is unlikely that the ortho and meta isomers of lc would coelute with lc in both GC and HPLC analysis.

(39) Singy, G. A.; Buchs, A. Helv. Chim. Acta 1974, 57, 1158-1169.

(40) Newcomb, M.; Manek, M. B.; Glenn, A. G. J. Am. Chem. Soc. 1991, 113, 949- 958.

(41) Newcomb, M.; Manek, M. B. J. Am. Chem. Soc. 1990, 112, 9662-9663.

(42) Newcomb, M.; Johnson, C. C.; Manek, M. B.; Varick, T. R. J. Am. Chem. Soc.

1992, 114, 10915-10921. (43) Shimoda, M.; Okura, I. J. Mol. Catal. 1992, 72, 263-267.

(44) Green, J.; Dalton, H. Biochem. J. 1989, 259, 167-172. 178

(45) Priestley, N. D.; Floss, H. G.; Froland, W. A.; Lipscomb, J. D.; Williams, P. G.; Morimoto, H. J. Am. Chem. Soc. 1992, 114, 7561-7562. (46) Howard, J. A.; Scaiano, J. C. Landolt-Bornstein Numerical Data and Functional Relationships in Scienceand Technology;Springer-Verlag: Berlin, 1984; Vol. 13, subvol. d, p 125-127. (47) Fox; B. G.; Froland, W. A.; Dege, J. E.; Lipscomb, J. D. J. Biol. Chem. 1989, 264, 10023-10033.

(48) Bollinger, J. M., Jr.; Edmondson, D. E.; Huynh, B. H.; Filley, J.; Norton, J.

R.; Stubbe, J. Science 1991, 253, 292-298.

(49) Kulmacz, R. J.; Ren, Y.; Tsai, A.-L.; Palmer, G. Biochemistry 1990, 29, 8760-

8771.

(50) Babcock, G. T.; El-Deeb, M. K.; Sandusky, P. O.; Whittaker, M. M.;

Whittaker, J. W. J. Am. Chem. Soc. 1992, 114, 3727-3734. (51) Magnus, K. A.; Hazes, B.; Ton-That, H.; Bonaventura, C.; Bonaventura, J.;

Hol, W. G. J. Proteins 1994, 19, 302-309.

(52) Ling, J.; Nestor, L. P.; Czernuszewicz, R. S.; Spiro, T. G.; Fraczkiewicz, R.;

Sharma, K. D.; Loehr, T. M.; Sanders-Loehr, J. J. Am. Chem. Soc. 1994, 116, 7682-

7691.

(53) Murch, B. P.; Bradley, F. C.; Que, L. J. J. Am. Chem. Soc. 1986, 108, 5027- 5028.

(54) Menage, S.; Brennan, B. A.; Juarez-Garcia, C.; Miinck, E.; Que, L., Jr. J. Am.

Chem. Soc. 1990, 112, 6423-6425.

(55) Kitajima, N.; Fukui, H.; Moro-oka, Y. J. Am. Chem. Soc. 1990, 112, 6402-

6403.

(56) Brennan, B. A.; Chen, Q.; Juarez-Garcia, C.; True, A. E.; O'Connor, C. J.; Que, L., Jr. Inorg. Chem. 1991, 30, 1937-1943. 179

(57) Kitajima, N.; Tamura, N.; Amagai, H.; Fukiu, H.; Moro-oka, Y.; Mizutani,

Y.; Kitagawa, T.; Mathur, R.; Heerwegh, K.; Reed, C. A.; Randall, C. R.; Que, L.,

Jr.; Tatsumi, K. J. Am. Chem. Soc. 1994, 116, 9071-9085.

(58) Dong, Y.; Menage, S.; Brennan, B. A.; Elgren, T. E.; Jang, H. G.; Pearce, L.

L.; Que, L., Jr. J. Am. Chem. Soc. 1993, 115, 1851-1859.

(59) Hayashi, Y.; Suzuki, M.; Uehara, A.; Mizutani, Y.; Kitagawa, T. Chemistry

Letters 1992, 91-94.

(60) Sawyer, D. T.; McDowell, M. S.; Spencer, L.; Tsang, P. K. S. Inorg. Chem. 1989, 28, 1166-1170.

(61) Tung, H.-C.; Kang, C.; Sawyer, D. T. J. Am. Chem. Soc. 1992, 114, 3445-3455.

(62) Barton, D. H. R.; Beviere, S. D.; Chavasiri, W.; Csuhai, E.; Doller, D.

Tetrahedron 1992, 48, 2895-2910.

(63) Leising, R. A.; Brennan, B. A.; Que, L., Jr. J. Am. Chem. Soc. 1991, 113, 3988-

3990.

(64) Liu, K. E.; Lippard, S. J., unpublished results.

(65) Eaton, P. E.; Yip, Y. C. J. Am. Chem. Soc. 1991, 113, 7692-7697.

(66) Lowry, T. H.; Richardson, K. S. Mechanism and Theroy in Organic Chemistry;

3rd ed.; Harper & Row: New York, 1987, p 222-227.

(67) Marcus, R. A. Ann. Rev. Phys. Chem. 1964, 15, 155. (68) Applications of Marcus theory to predict the rate constants of group transfer reactions of radicals are generally quite accurate when charge transfer states are unimportant in the radical reactions. 69 A Marcus approach was used successfully to estimate rate constants for hydrogen atom transfer reactions.40 . (69) Fox, G. L.; Schlegel, H. B. J. Phys. Chem. 1992, 96, 298-302. (70) Maillard, B.; Forrest, D.; Ingold, K. U. J. Am. Chem. Soc. 1976, 98, 7024-7026. (71) Mathew, L.; Warkentin, J. J. Am. Chem. Soc. 1986, 108, 7981-7984.

(72) Newcomb, M.; Glenn, A. G. J. Am. Chem. Soc. 1989, 111, 275-277. 180

(73) Bechwith, A. L. J.; Bowry, V. W.; Moad, G. J. Org. Chem. 1988, 53, 1632-

1641. (74) The reported7 5 rate constant for cyclization of the 3-butenyl radical has been corrected using a more recently determined value76 for the rate constant for reaction of Bu3SnH with a primary radical. (75) Effio, A.; Griller, D.; Ingold, K. U.; Beckwith, A. L. J.; Serelis, A. K. J. Am.

Chem. Soc. 1980, 102, 1734-1736.

(76) Johnston, L. J.; Lusztyk, J.; Wayner, D. D. M.; Abeywickreyma, A. N.; Beckwith, A. L. J.; Scaiano, J. C.; Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 4594- 4596.

1(77) McMillen, D. F.; Golden, D. M. Ann. Rev. Phys. Chem. 1982, 33, 493-531.

(78) Bordwell, F. G.; Cheng, J.-P.; Harrelson, J. A., Jr. J. Am. Chem. Soc. 1988,

::Z10,1229-1231. (79) For a related treatment of stereoelectronic effects on the kinetics of radical reactions, see Tanko, J. M.; Kamrudin, N.; Blackert, J. F. J. Org. Chem. 1991, 56, 6395-6399. (80) Another possible explanation for formation of cyclic alcohol la, reversible ring opening of the radical from 1 and selective trapping of the cyclic radical, can be excluded readily. From the expected exergonicity of the ring opening reaction (i.e. AG° = - 18.2 kcal/mol), the equilibrium constant for the ring opening at room temperature is about 2 x 1013. With a ring opening rate constant of 3 x 1011 s-1, the ring closure of the acyclic radical must have a rate constant on the order of 0.01 s-1 . That the benzylic radical could have a lifetime of 100 s at room temperature is without precedence, and the turnover rate of MMO exceeds this value.

(81) Martin-Esker, A. A.; Johnson, C. C.; Horner, J. H.; Newcomb, M. J. Am. Chem. Soc. 1994, 116, 9174-9181. 181

(82) O'Ferrall, R. A. M. J. Chem. Soc. (B) 1970, 785-790.

(83) Adam, W.; Curci, R.; Edwards, J. O. Acc. Chem. Res. 1989, 22, 205-211.

(84) Murray, R. W.; Jeyaraman, R.; Mohan, L. J. Am. Chem. Soc. 1986, 108, 2470- 2472.

(85) Tsuda, M.; Oikawa, S.; Okamura, Y.; Kimura, K.; Urabe, T.; Nakajima, M.

Chem. Pharm. Bull. 1986, 34, 4457-4466. (86) Jiang, Y.; Wilkins, P. C.; Dalton, H. Biochim. Biophys. Acta 1993, 1163, 105-

112. (87) Groves, J. T.; McClusky, G. A. Biochemical and Biophysical Research

Communications 1978, 81, 154-160.

(88) Groves, J. T.; Subramanian, D. V. J. Am. Chem. Soc. 1984, 106, 2177-2181.

(89) Groves, J. T.; Avaria-Neisser, G. E.; Fish, K. M.; Imachi, M.; Kuczkowski,

R. L. J. Am. Chem. Soc. 1986, 108, 3837-3838.

(90) Rosenzweig, A. C.; Feng, X.; Lippard, S. J. In IUCCP Symposium on Applications of Enzyme BiotechnologyPlenum Press: College Station, TX, 1991; pp

182-203.

(91) Tsien, H.-C.; Hanson, R. S. Appl. Environ. Microbiol. 1992, 58, 953-960. 182

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Table 3. Expected Products and Observed Ratios for Reactions with MMO from A. capsulatus (Bath). Rearranged Observed Ratio of Rate Constant Unrearranged Alcohol(s) Unrearranged to For Radical Rear- Substrate Product(s) Expected Rerranged Alcohol rangement, s -1 at 45 °C OH >100:1 4 x 101' PhPhi. OH Phib 1 la lb

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CHAPTER 5.

Tritiated Chiral Alkanes as

Substrates for Methane Monooxygenase from Methylococcus capsulatus (Bath): Probes for the Mechansim of Hydroxylation 211 Preface. This chapter describes work which is ongoing at the time of this writing. Most of the results are preliminary, and the following discussion should not be taken to represent the final conclusions from this series of experiments. In particular, the exchange process currently remains under investigation. The work described here was carried out with pure hydroxylase and an unresolved mixture of protein B and reductase. Direct evidence for an exchange process was obtained when a significant amount of

CD3CDHOH was recovered from reactions with C2D6. We have ruled out the possibility that the exchange arises from an artifactual property of the unresolved mixture of protein B and reductase since no scrambling of

CD3CD20H takes place in the absence of pure hydroxylase (see discussion). Introduction. The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b rely on methane monooxygenase (MMO) to convert methane into methanol in the first step of their biosynthetic pathways. 1 This reaction provides the organisms with their sole source of carbon and energy according to equation (1). MMO exists in both a particulate and a soluble form.1 The former resides in the cell membranes under normal

+ + CH4 + 02 + NADH + H - CH30H + H20 + NAD (1) growth conditions and contains copper at its active site.2 Under low copper concentrations, the methanotroph switches on the synthesis of the soluble MMO. 3 Soluble MMO's from both Methylococcus capsulatus (Bath)4 and Methylosinus trichosporiumOB3b 5 comprise three proteins, a hydroxylase component (MMOH) that binds the hydrocarbon substrate and dioxygen,6 a 212 reductase (MMOR) containing Fe2S2 and FAD cofactors which enable it to accept electrons from NADH and transfer them to the hydroxylase,4' 7 and a third component, protein B (MMOB), which regulates electron transfer between the reductase and hydroxylase.8 In addition to methane, the MMO system is capable of oxidizing a wide variety of substrates.9 -11 X-ray crystallographic studies of the resting, diferric form of the hydroxylase, 12 ENDOR spectroscopy of the mixed-valent state,13 and other physical methods 6'1 4 indicate that the M. capsulatus (Bath) MMOH active site contains a non-heme dinuclear iron center, the structure of which is given on the following page. As shown in this drawing, the iron atoms can be additionally bridged by an exogenous acetate ligand introduced under the crystallization conditions for the x-ray structural investigation.12

CH3 acetate water O l o Glu 243

Glu 114 Glu 209 Fe, Fe 2 Glu 209

N H N His 147 0 0 His 246

Glu 144

The heme enzyme cytochrome P-450 displays similar reactivity to MMO with the exception that it will not hydroxylate methane.15 Cytochrome P-450 oxidations are thought to proceed through a high valent iron oxo, or ferryl, intermediate. 15 -18 This species is postulated to abstract a hydrogen atom from the hydrocarbon substrate, resulting in a substrate radical and a coordinated hydroxyl radical. The hydroxyl radical and substrate radical next recombine in a "rebound" reaction to afford the product alcohol, which disso- ciates from the active site leaving the resting state of the enzyme. Radical clock probes which rearrange upon formation of a substrate radical have been 213 useful in establishing the presence of substrate radicals in the reaction cycle of cytochrome P-450.19 -21 The radical clock results reported in Chapter 4 differ for MMO systems isolated from the two organisms M. capsulatus (Bath) and M. trichosporium OB3b. With M. capsulatus (Bath), no evidence for the formation of substrate radicals was found with all substrates tested. Based on the known rate constants for rearrangement of the various probes, the lower limit for a rebound reaction was estimated to be 1013 s-1 at 45 °C. For M. trichosporium OB3b, however, evidence for substrate radicals was detected in the form of rearranged product alcohols. Based on the amount of rearrangement observed, a rate constant of 6 to 9 x 1012 s- l at 30 C was estimated for a rebound reaction with this enzyme. Evidence for intermediate radical formation has also been obtained in other studies with MMO from M. trichosporium OB3b,2 2-24 including a report with the substrates (R)-[1-2H,1-3H]-ethane and (S)-[1-2H,1-3H]-ethane. These results were interpreted as evidence that the hydroxylation reaction with these substrates proceeds through a mechanism analogous to that widely accepted for cytochrome P-450 hydroxylation, as illustrated in Figure 1. Upon abstraction of a hydrogen atom from the chiral substrate, the ethyl radical results. This substrate radical has the option to rebound and form the unrearranged product alcohol. Alternatively, since the carbon radical is now planar, rotation about the C-C bond prior to recombination in the rebound reaction leads to a rearranged product alcohol with the opposite configuration. Both alcohol products can then be derivatized with a chiral acid, and 3H NMR analysis of the diastereomeric esters used to quantitate the amount of retention or inversion that occurred during the hydroxylation reaction. Results with MMO from M. trichosporium OB3b indicated that, for 214 both R and S chiral ethane substrates, = 65 % of the product alcohols displayed retention whereas = 35 % displayed inversion.2 2 Based on a value of 0.5 kcal for rotation about the C-C bond, a rate constant of 3 x 1012 -1 can be calculated for the rebound reaction rate constant for the substrate radical. This value agrees well with that estimated from the radical clock work.25 Although the radical clock substrate probes, which displayed slightly different reactivity with MMO from the different organisms, gave results in agreement with the chiral ethane experiment, we were interested in determining whether the differences between the M. capuslatus (Bath) and M. trichosporiumOB3b enzymes would persist with a substrate purported more representative 22 of methane. Accordingly, experiments employing the chiral ethane methodology were carried out with MMO from M. capsulatus (Bath) in collaboration with Dr Philip Williams and Dr. Hiromi Morimoto at the National Tritium Labeling Facility and with Professor Heinz Floss and Dr. Barrie Wilkinson at the University of Washington. This group of collaborators is the same who participated in studies with the M. trichosporium OB3b MMO. Accordingly, the substrates (S)-[1-2 H,1-3 H]-ethane, (R)-[1-2H,1-3H]-ethane, (S)-[1-2H,1-3H]-butane, (R)-[1-2 H,1-3H]-butane, (S)-[2- 3 H]-butane and (R)-[2-3H]-ethane were hydroxylated with MMO from M. capsulatus (Bath) and the results and analysis are reported here. Experimental. Synthesis of the substrate precursors was performed by Dr. Barrie Wilkinson. NMR data were collected by Dr. Philip Williams at NTLF. Bacterial Growth and Protein Purification. Methylococcus capsulatus (Bath) cells were grown as described previously. 26 Hydroxylase was purified as reported elsewhere, and the iron content and specific activities were in the reported ranges.1 4 '25 In the 215 enzymatic reactions, an unresolved mixture of protein B and reductase from the DEAE cellulose column in the hydroxylase purification method was added to the hydroxylase. The ratio of this protein B-reductase mixture relative to hydroxylase was determined by maximizing hydroxylase activity with propylene as substrate. Preliminary assays with unlabelled (proteo) ethane and butane were carried out to determine conditions under which 1 to 2 mmole of product alcohol could be produced. MMOH was concentrated to = 300 gM and incubated with the MMOB-MMORmixture in a 5 ml reaction flask which was capped with a septum. The resulting volume was adjusted to 400 Rl with 25 mM MOPS, pH 7.0 buffer. A syringe was inserted through the septum and 2 ml of the head space gas was removed and replaced with 2 ml of the substrate gas. The mixture was incubated for 30 s at 45 C in a shaking incubator, after which time 100 Rl of a 0.1 M ethanol-free solution of NADH was added to initiate the reaction. The flask was returned to the incubator and the reaction was allowed to proceed for 5 min. A 5 Rl portion of the solution was next injected into a GC equipped with a Porapak Q column to quantitate the concentration of alcohol product present in the reaction solution. The temperature settings were 200 °C for the injector, 225 °C for the FID detector, and 180 °C for the oven. The area of the product peak was then compared to a previously constructed calibration curve of alcohol peak area vs the concentration of sample injected. Conditions which afforded at least 1 mmol of alcohol product per 1 ml of reaction volume was produced were subsequently employed at NTLF. Under these conditions, the specific activity calculated for the hydroxylase is low, 40 mU/mg, owing to product inhibition. Quantities of the proteins used in various runs at NTLF are listed in Table 1. 216 Reagent Synthesis. 2 7 (S)-[1-1H,1-2H]-Ethanol. [1-2H]-Acetaldehyde (0.56 ml, 10 mmole) was added to a stirred solution of (R)-alpine hydride (10 mmole) in 20 ml dry THF at -70 °C under argon. The temperature was maintained at -70 °C for 4 h, after which time the mixture was allowed to warm to room temperature. After 15 to 18 h, the solution was cooled to 0 C and ethanolamine (0.74 ml, 12.3 mmole) was added. After 30 min a white precipitate formed. The solvent was removed by distillation, during which the precipitate dissolved upon warming. Just before the end of the distillation, 5 ml of benzene was added to, and distilled from, the mixture. The benzene addition and distillation steps were repeated twice. The final volume of the collected distillates was 32 ml. (R)-[1-1H,1-2H]-Ethanol , (S)-[1-1H,1-2H]-butanol, (R)-[1-1H,1-2 H]-butanol, (S)-2-butanol, and (R)-2-butanol were synthesized by using similar procedures. The purity of these materials was >95% as judged by 1H NMR spectroscopy. (S)-[1-1H,1-2H]-Ethyl Tosylate. (S)-[1-1H,1-2H]-Ethanol (16 mmole in THF) was added to a stirred solution of tosyl chloride (3.8 g, 20 mmole, freshly crystallized from hexane) in dry pyridine (15 ml, distilled from KOH under argon) under argon at room temperature. Pyridine HCl began to precipitate almost immediately. The reaction was allowed to continue for 5 h, after which time it was terminated by the addition of 100 ml of diethyl ether and

100 ml of a saturated aqueous solution of CuSO4. The organic phase was

collected and washed with two 50 ml portions of saturated CuSO4, 30 ml of a 1

N HCl solution, and 50 ml of a saturated aqueous solution of NaHCO 3. The

organic phase was dried over Na2SO4 and filtered to remove the solid. The cdesired product was isolated by silica gel column chromatography and eluted with 7 % diethyl ether in hexane. (R)-[l-1H,1- 2H]-Ethyl tosylate, (S)-[1-1H,- 217 2H]-butyl tosylate, (R)-[1-1H,1-2H]-butyl tosylate, (S)-2--butanyl tosylate, and (R)-2-butyl tosylate were synthesized following analogous methods. The purity of were judged by NMR spctroscopy >95%. (S)-[1-2H,1-3H]-Ethane. A diagram of the reaction apparatus is shown 3 28 in Figure 2. The reagent LiEt3B H was prepared in the following manner. 3 H2 ( 100 Ci) was first transferred from a uranium tritide bed thermostatted at 350 C to a 10 ml round bottom flask fitted with a stopcock (A in Figure 2) and septum-capped sidearm at a final pressure of 625 mm Hg. By means of a gas-tight syringe, n-BuLi (166 gl dissolved in hexanes, 0.027 mmole) and TMEDA2 9 (34 pl, 0.23 mmole) were added to the flask through the septum. The resulting mixture was stirred at room temperature for 45 min, during which time a white, creamy precipitate (Li3H) formed. The round bottom flask was evacuated to dryness, reaching a pressure of = 30 igm Hg. THF (100 gl) and dinitrogen were added to a final pressure of 150 jgm Hg. The resulting 3 Li H slurry was treated with Et3B (200 gl in a THF solution, 0.2 mmol) to dissolve the Li3H. The alkyl tosylate (= 0.3 mmole), dissolved in 100 1 of THF, was added to the flask and vigorous gas evolution occurred for 15 to 20 s. After a 1 h incubation period, the flask was cooled to -78 C. Stopcock C was opened and the gas was transferred to a coconut charcoal bed (200 mg) in a test tube immersed in liquid nitrogen. The transfer was allowed to occur for 60 s. Identical procedures were used to synthesize (R)-[1-2 H,1-3H]-ethane, (S)-[1- 2 3 2 3 3 3 H,1- H]-butane, (R)-1-[ Hl, 1 - H 1]-butane, (S)-[2- H]-butane, and (R)-[2- H]- butane from their corresponding alkyl tosylates. Purity levels >95% were estimated by 1 H NMR. Enzymatic Reactions. A second round bottom flask equipped with a stopcock, sidearm, and septum was fitted to the reaction apparatus (B in Figure 2) and evacuated. 218 Buffer (25 mM MOPS, pH 7.0, 100 to 500 gl) was added through the septum and cooled to -78 C. Substrate gas was transferred from the charcoal bed by opening stopcock C and stopcock B on the reaction flask. The bed was brought to room temperature by warming with hot air during this procedure. After 60 s the stopcocks were closed and the buffer was brought to room temperature. The protein solution was next added through the septum. A quantity of NADH (50 or 100 gl, see Table 1) was also added. Pure 02 gas (1 ml) was added by syringe through the septum and the mixture was incubated at 45 + 2 °C with constant stirring for 30 min. Cooling the flask to - 78 C terminated the reaction. Excess substrate gas was transferred from the reaction flask to the charcoal bed by first cooling the bed to = 77 K and then opening stopcocks B and C. Transfer was allowed to proceed for 60 s and then the stopcocks were closed. A small amount (10 l) of the expected product alcohol (unlabeled) was added to the reaction flask to act as a carrier in subsequent manipulations. This flask was removed from the apparatus and transferred to a vacuum where the volatile reaction products were collected by lyophilization. Derivitization. 30 (R)-O-Acetyl mandelic acid (47 mg, 0.24 mmol) and DMAP (2 mg) were mixed in 2 ml of methylene chloride at -40 C in a 100 ml round bottom flask. Over a 5 min period, a solution of DCCI (54 mg, 0.24 mmole) in 0.5 ml methylene chloride was added dropwise to the flask. A creamy precipitate resulted after 10 min. The enzymatic lypholysate was added dropwise over a 5 min period. The resulting mixture was allowed to warm to room temperature over 3 to 6 h and then stirred overnight. The suspension was filtered through a silica plug ( 2 g) and washed with 35 to 40 ml of methylene chloride. 219 The filtrate was next evaporated to near dryness under a dinitrogen stream and then lypholized to remove any methylene chloride. The residue was suspended in d6-benzene and filtered through a glass wool plug directly into an NMR tube. Product Analysis. To measure the radioactivity of the samples, a 1 gl aliquot of the product solution was diluted in 200 l1of methanol before being placed in a liquid scintillation counter. NMR analysis was carried out with a 300 MHz Bruker spectrometer at NTLF. All 3H spectra were 1H decoupled. Results. Analysis of Lypholysates. The possible alcohol products of the reaction of (S)-[1-2H,1-3 H]-ethane with MMO are depicted in Figure 3. Their actual 1H and 3H NMR spectra are given in Figures 4 and 5, respectively. The 1H spectrum shows a large water peak at 4.82 ppm. Since only = 1 mole of product was formed (mM ethanol expected in 1 ml total volume), the carrier alcohol dominates the 1H spectrum. Accordingly, the signals at 6 = 1.20 ppm (t, J = 6 Hz, 3H) and 6 = 3.67 ppm (q, J = 6 Hz, 2H) were observed for ethanol. A minor signal at 6 = 3.38 ppm is currently unassigned. The 3H NMR spectrum of the reaction products in Figure 5 contains four major resonances at 6 = 1.19 (s), 6 = 3.65 (s), 6 = 3.67 (s), and 6 = 4.84 (s) ppm. The resonance at = 4.84 ppm was assigned to H3HO, and the singlet at = 1.19 ppm to the product of oxidation occurring at the unlabeled carbon atom (5 in Figure 3). The inset in Figure 5 shows an expansion of the two signals at = 3.65 and 3.67 ppm. The narrower, more downfield resonance is attributed to 1 and 4 (H,T products), whereas the broader, more upfield signal is assigned as arising from 2 and 3 (D,T products). The broadening of the latter signal is due to unresolved coupling to the deuterium atom present in 220 the molecule. A kinetic isotope effect can be calculated from the ratio of alcohol products for hydroxylation of the C-H bond (2 and 3) versus products hydroxylated at the C-D bond (1 and 4). Taking the ratio of integrals at 3.65 and 3.67 ppm gives a kH/kD value of 0.7, which should be considered approximate since the peaks are not baseline resolved. The sum of the areas of these two peaks can be compared to the area at = 1.19 ppm to obtain information about the relative hydroxylation rates at the two different carbon atoms. The observed integrations indicate that oxidation at the unlabelled carbon atom was favored by a factor of 3 over oxidation at both the proton and deuteron of the labeled carbon. This value may reflect the relative reactivities of the C-H versus C-D bonds under the assumption that once bound to the active site, ethane is free to rotate and present either of its carbon atoms to the active hydroxylating species. Reaction at the tritium atom will not be revealed in the 3H NMR spectrum, but is expected to be very slow. These experiments were carried out under Vmax conditions, in which a saturating amount of substrate was added to the enzyme, which could lead to an artificially low intermolecular isotope effects, however. Analysis of Derivatives. The derivitization reaction, illustrated in Figure 6,30 can afford five 3H NMR resonances, two sets arising from diastereomers and one from another product. These peaks are shown in Figures 7 (whole spectrum) and 8 (expanded version). The products arising from stereochemical inversion are labeled A and D, those with retention are labeled B and C. The loss of stereochemistry, calculated by adding the areas of peaks A and D and dividing by the combined areas of all four peaks, was 14.5 /o (85.5% retention). The values corresponding to products obtained by hydroxylation at the C-H bonds versus hydroxylation of the C-D bonds were 221 39%/0(69 % retention) and 7% (93 % retention), respectively. The reasons for separating the products in this manner will become clear shortly. The 3H NMR spectrum of the alcohol products formed by hydroxylation of (S)-[1-2H,1-3H]-butane with MMO is given in Figure 9. The spectra of corresponding mandelate derivatives are given in Figures 10 (whole spectrum) and 11 (expanded version). The 3H spectrum of the mandelate derivatives of alcohols formed with (S)-[2-3H]-butane is given in Figure 12, and the spectrum of derivatized products formed by reactions with a racemic mixture of this substrate is shown in Figure 13. All assignments are indicated on the spectra.27 Table 2 summarizes the results for two trials of the ethane hydroxylations, Table 3 reports results for reactions with (R) and (S)-[1- 2H,1-3H]-butane, and Table 4 lists the findings for butane substrates labeled at the C2 position. When the results in Tables 2 and 3 are analyzed according to the several categories of C-H bond hydroxylation, several interesting clues about the hydroxylation mechanism of MMO emerge. For each ethane enantiomer hydroxylation can take place either at the hydrogen atom (D,T products) or with the deuterium atom (H,T products) of the chiral carbon. Products that arise from hydroxylation of the C-T bond are not detected since 3H NMR is the analytical method. From the relative amounts of C-H versus C-D hydroxylation products were calculated the intramolecular kinetic isotope effects, kH/kD (Tables 2 and 3). The 3H NMR data also afford information about the distribution of hydroxylation at each carbon atom of the substrate. These ratios of products, C2/C1 and C4/C1 for ethane and butane, respectively are also included in the Tables.

As indicated in Table 2, hydroxylation of (R)-[1-2 H,1- 3 H]-ethane and (S)- [1-2H,1- 3H]-ethane gave very different results. With (R)-[1-2H,1-3H]-ethane, 222 the kinetic isotope effect calculated from the 3H NMR of the alcohol products was kH/kD = 1.1. This value agrees with that obtained (0.9) from adding the integrals of peaks B and D and dividing by the sum of A and C in the spectrum of the mandelate derivative, shown in Figure 8. The corresponding numbers for (S)-[1-2 H,1-3H]-ethane hydroxylation differ, however. From the alcohol products, kH/kD = 0.2 and 0.7, in two runs, whereas from the derivatives, values of 0.5 and 0.1 were obtained. Large ratios of products formed by hydroxylation at C2 vs C1 ranging from 3.5 to 4.7, were observed with both enantiomers. The amount of inversion for the (R) and (S)-[1-2 H,1-3 H]-ethanes also varied. For (R)-[1-2 H,1-3H]-ethane, almost racemic and even inverted mixtures of the derivatives were observed, as indicated in Table 2. Products arising from hydroxylation of the C-H bond (D,T products), however, showed 83 and 77 % retention. Reaction at the C-D bond (H,T products) of (R)-[1-2H,1- 3H]-ethane gave predominantly inverted products, 80 and 93% inversion. These latter values skew the summed (D,T and H,T) distribution of products such that they appear to be almost racemic. For (S)-[1-2 H,1- 3 H]-ethane, different behavior is again observed (Table 2). Analysis of the D,T products reveals 69 to 100 % retention, and the H,T products show 68 to 93% retention. This strange behavior is also reflected in the results for MMO hydroxylation of (R)-[1-2 H,1-3H]-butane and (S)-[1-2 H,1-3 H]-butane (Table 3). A kinetic isotope effect of kH/kD = 1 was computed for (S)-[1- 2 H,1- 3 H]-butane hydroxylation, whereas a value of < 0.2 was calculated from the alcohol and derivative spectra for hydroxylation with (R)-[1-2 H,1-3 H]-butane. The stereochemical results for the D,T products showed 66 % retention with (R)- [1-2H,1-3H]-butane and 51 % retention with (S)-[1-2 H,1-3H]-butane. The H,T 223 alcohols gave 93 % inversion with (R)-[1-2H,1-3H]-butane and 51 % retention with (S)-[1-2H,1-3H]-butane. Retention of stereochemistry was primarily seen with both (R)-[2-3H]- butane and (S)-[2-3H]-butane (Table 4). Products obtained from a racemic mixture of (R)-[2-3H]-butane and (S)-[2-3H]-butane showed only a slight preference for hydroxylation of one enantiomer over the other (Figure 13 and Table 4). The product ratios were 1: 1.4. More interestingly, oxidation at the C-3 position, which leads to diastereotopic products, proceeded in a stereoselective manner. One product was formed in 2.5 fold excess over the other, as shown in Figure 14. Discussion. Analysis of Product Distributions. Alkanes Are Not Highly Constricted in the Active Site Prior to Hydroxylation. The reaction products obtained with racemic [2-3 H]-butane indicate clearly that the active site does not rigorously constrain these substrates, since there was only a slight discrimination between the two enantiomers of this probe. An extremely crowded active site would most likely readily distinguish an ethyl and a methyl substituent at the reacting carbon atom. The products of oxidation at the C3 position using racemic substrate, however, show some stereoselectivity. Two diastereotopic products were observed, the ratio of one to the other as detected by 3H NMR spectroscopy being 2.5: 1. This behavior indicates that there is some degree of chiral discrimination conferred by the active site, but at the 3- rather than the a-carbon. One possible explanation for the different product alcohol stereochemistries observed for hydroxylation of the two enantiomers of [1- 2H,3 H]-ethane or [1-2 H,3 H]-butane is that the substrates are stereochemically 224 cdistinguished in binding to the active site of the hydroxylase. For example, the (R) enantiomer of [1-2 H,3H]-ethane might be specifically oriented in such a manner that chiral discrimination between the tritium and deuterium groups could take place prior to C-H bond hydroxylation. Such an effect would require restricted orientation of the methyl group with respect to the diiron center. Similar chiral discrimination was seen with the much bulkier radical clock substrate probe trans-2-phenylmethylcyclopropane, 25 as described in Chapter 4. A example of how such substrate binding prior to hydroxylation by MMO might be manifest is given in Figure 15. If such a chiral discrimination were taking place to account for the differences in the product distribution between the (R) and (S) enantiomers of [1-2H,3 H]-ethane, one would expect the butane enantiomers to behave similarly, as illustrated for (R)-[1-2H,1-3H]- butane in Figure 16. The methyl, deuterium, and tritium substituents of the reacting carbon for chiral ethane would be expected to bind in the same positions as the analogous groups of the reacting carbon atom of butane in such a tightly constrained site. Accordingly, similar alcohol product stereochemistries would be expected for chiral butane as for chiral ethane. The total (R) and (S) butane products do not, however, follow similar stereochemical patterns, (cf. Tables 2 and 3) suggesting that this explanation is not viable, in agreement with our interpretation of the [2-3H]-butane results. H,T vs D,T Product Stereochemistries and Hydrogen Exchange at the a- Carbon of the Product Alcohols. Although the total amounts of retention and inversion for the two enantiomers of chiral ethane versus chiral butane are very different, further analysis reveals interesting trends in the data. The (R) probes lead predominantly to retention when the reaction yields D,T products and to inversion for most of the alcohols in the H,T pool. With the 225 (S) ethane probes, predominant retention occurs with both D,T and H,T products. For (S) butane products, however, the products are nearly racemic in both the D,T and H,T pools. Although the extent of retention in the H,T and D,T product pools for each substrate enantiomer was initially confusing, it can be rationalized by postulating the occurrence of an exchange process in the product alcohols which masks the true stereochemistry of the products formed in the hydroxylation reactions. In this model, hydroxylation occurs leaving the product molecule bound to one or both (Figure 17) iron atoms in the active site. Alternatively, this same species could arise from binding of the released product alcohol to a different hydroxylase molecule. A reaction in which a protein residue exchanges hydrogen with the bound product in a stereospecific manner might then occur. This exchange reaction must take place stereospecifically in order to account for the observed results. Both the extent of inversion for each enantiomer, and the composition of the H,T and D,T product pools, can be rationalized by the extent to which such an exchange process occurs. As indicated below, the observed product distributions can be accounted for by the relative binding affinities of the product alcohols (vide infra). Product binding to the oxidized diiron site has been demonstrated for phenol with hydroxylase from M. trichosporium OB3b.31 An exchange process has been suggested following hydroxylations by cytochrome P-450.3 2 Stereochemical scrambling of product alcohols which have fully retained or fully inverted their stereochemistry can be explained by such an exchange mechanism. Figures 18 and 19 show the effects of exchange on products formed with retention and inversion for the substrate (R)-[1-2 H,1- 3H]-ethane. Exchange of hydrogen in a stereospecific manner would lead to 226 the following results. Consider the H,T (Figure 18) and D,T (Figure 19) products formed initially. For one H,T product, exchange leaves the product stereochemistry unmodified. For the other, tritium is lost in the exchange and the product is therefore not observed. Exchange of one D,T product similarly leads to an unobserved alcohol. The second D,T product, however, is converted to an H,T product of the same configuration upon exchange! This H,T product signifies an apparent inversion of configuration, even though the hydroxylation step occurred with retention. This last case demonstrates how a stereospecific exchange process can lead to erroneous conclusions about the nature of the hydroxylation step. The quantitative effects of such an exchange mechanism on the product stereochemistries as well as on both the intramolecular kinetic isotope effects (kH/kD, at C1) and C2/C1 product ratios can be computed. In Table 5, four sets of such calculations are presented. In case A, hydroxylation of (R)-[1-2H,1-3 H]-ethane is considered to occur with complete racemization and an intramolecular kinetic isotope effect of kH/kD = 4. The corresponding amounts of species 1 through 4 are listed for no subsequent exchange, with 50% exchange, and with 75% exchange of the product alcohols. The effects of these exchange reactions on the various categories of H,T products, D,T products, C2/C1 ratio, and kH/kD are also tabulated. A second set of calculations was performed (case B) for the same substrate and again taking an intial kH/kD value of 4, but assuming that the hydroxylation step occurs with 33% inversion.. The last two sets of calculations were made with (S)-[1-2H,1- 3H]-ethane and the same initial assumtions (cases C and D). These calculations illustrate how product exchange can skew the distribution of products and lead to the observed stereochemistry. The effects on the kinetic isotope effects are discussed below. 227 The low specific activity ( 40 mU/mg) observed for the hydroxylase under conditions required to produce the large quantities of product needed for 3H NMR studies, = 1 mmole/1 ml, compared to the = 0.1 mmole/1 ml, amount used under normal assay conditions, where SPA = 250 mU/mg, suggests strong inhibition of the enzyme by the product alcohols. These species have a significant binding affinity for the active site which enables them to compete with substrate. Since the product alcohol binds well, it is reasonable to suggest that it may bind with a long enough lifetime for an exchange reaction to take place. No evidence for exchange was suggested in the results for the radical clock product alcohols, which probably reflects their relatively poor binding affinity at the active site. On the other hand, very 33 recent results from our laboratory have revealed that, when C2D6 is used as

substrate, a substantial amount of C2D4HOH forms. Control reactions in which hydroxylase was omitted from the reaction mixture failed to show

exchange of CH3CD2OH. Thus an exchange mechanism such as that depicted in Figures 18 and 19 appears to occur, and is dependent on the presence of the hydroxylase component. Kinetic Isotope Effects. Figure 20 illustrates how the C2/C1 hydroxylation can depend upon the magnitude of the intramolecular kinetic isotope effect at C1, referred to as kH/kD. The ability of product exchange to influence both the C2/C1 and kH/kD values from a reaction with (S)-[1-2 H,1-3H]-ethane is presented in Figure 21. Products arising from hydroxylation at the C-H bond are designated as "kH" alcohols, whereas reactions at the C-D bond are labeled "kD" alcohols. For both "kH" and "kD" alcohols, the tritium label can be lost, affording undetectable products. Exchange of "kD" alcohols at their C-H bond will have no effect since they are replaced with C-H bonds. Exchange of "kH" 228 products, however, will convert C-D bonds into C-H bonds, resulting in species scored as "kD" products. Exchange thus depletes "kH" products while increasing the pool of "kD" alcohols, resulting in a decrease in the apparent kH/kD value. The exchange process will also decrease C2/C1 in the following manner. The sum of "kH" and "kD" alcohols represents C1 products. Both sets of these alcohols lose tritium upon exchange (Figure 21) depleting the pool of C1 products detected. Exchange will have no effect on C2 products, since this carbon only has C-H bonds and the label at the second carbon atom will remain intact. Since C1 is diminished and C2 was left unaffected, the value of C2/C1 will increase after exchange. The calculations presented in Table 5 quantitatively show how the exchange process can artificially decrease the measured kH/kD, while simultaneously increasing C2/C1 from predicted values with a given kH/kD for the primary hydroxylation step. Another factor that may contribute to the high C2/C1 value should be considered, however. It is possible that the hydroxylating species in MMOH has equal access to hydrogen atoms on both the C1 and C2 positions of chiral ethane. In this case, the ratio of C2/C1 products would reflect the relative ability to hydroxylate C-H versus C-D bonds, as for an intramolecular isotope effect. Previous studies comparing R-CH3 with R-CD3 alkanes have yielded intermolecular kinetic isotope effects of = 1.25,34 The substrates employed in these studies were significantly larger than ethane or butane, and contained either a tertiary or quaternary carbon atom to the reacting methyl group. Such highly substituted carbons react much more slowly with MMO than primary C-H groups, and products corresponding to reaction at the a position were not detected. It is possible that, with chiral ethane, rotation occurs in the active site rendering both ends of the molecule accessible to the active hydroxylating species. With chiral butane, reaction at C4 instead of C1 would 229 occur less often than the reaction of C2 versus C1 in ethane, accounting for the lower values of C4/C1, = 3, compared to C2/C1 values = 3.5 to 4.4 for the ethanes. Preorientation of the substrate at the reactive iron center may also affect kH/kD. For example, the C-H bond of the reacting carbon atom might interact with the reactive hydroxylating species preferentially over a C-D or a C-T bond. Mechanism A in Figure 22 suggests one such alignment for an agostic interaction with the Fe atom adjacent to a ferryl species. Orientation of the C-H bond in this manner exposes the C-D or C-T bonds preferentially for the hydroxylation reaction. Such behavior could account for the inverse isotope effect observed in some cases, but does not account for the different D,T and H,T products stereochemistries. Secondary kinetic isotope effects from a substituent adjacent to the reacting atom in the hydroxylation step could also play some role in determining the stereochemistry of the product, but these effects are expected to be very small. Mechanistic Implications. We now inquire whether, given the foregoing analysis, the experimental results can be accounted for if the hydroxylation step were to occur with complete retention or complete inversion of stereochemistry followed by exchange of protium for protium, deuterium, or tritium, as indicated in Figures 18 and 19. Even for cases where retention or inversion is extremely dominant, such as > 90 % retention, an exchange mechanism alone is not sufficient to explain all of the measured product distributions. Figure 23 shows the four tritiated alcohols that can be observed by 3H NMR spectroscopy. Products C and D represent inverted species. If the alcohol were formed initially with 100 % retention, meaning that only A and B are produced, then conversion of these products to C and D must occur. Given 230 the restrictions on the exchange process outlined above, no pathway for conversion of A or B into D can be found. Since this product is detected in the 3H NMR spectrum of the derivative made from the product of (S)-[1-2H,1- 3H]-ethane hydroxylation, it must be present in the product alcohols. All manipulations, including the derivitization procedures, are not expected scramble the stereochemistry.27 Given the above analysis, it seems likely that there exists some radical or possibly carbocation character in the hydroxylation step to account for all the observed products. The extent of such involvement seems to vary between enantiomers of a given substrate and even between the substrates themselves, as reflected in the different stereochemical results. This observation has several implications. There could be two parallel reaction pathways, one involving a substrate radical and one not. Two possible pathways are presented in Figure 22. The degree to which a radical vs. non- radical mechanism is followed may be determined by how snugly the substrate fits into the active site. Parallel mechanistic pathways have similarly been invoked in porphyrin model systems to explain the stereochemical consequences of hydroxylation chemistry depending on steric interactions of the substrate with the catalyst.3 5 -3 7 Alternatively, abstraction of a hydrogen atom to form a substrate radical may occur in every reaction. The rate constant for a rebound reaction, such as that presented in mechanism B of Figure 22, may be so large that only a small quantity of the substrate radical has a sufficient lifetime for rotation about the C-C bond to occur before recombination with the bound hydroxyl group. Results with radical clock substrate probes were consistent with either no substrate radical formation or an extremely fast rebound step having a rate constant on the order of 1013 s1. A rebound rate constant in the experiments reported here 231 cannot be accurately estimated since the occurrence of exchange distorts the distribution of products formed in the actual hydroxylation step. Comparisons with Literature. Very different stereochemical behavior was observed in the present experiments with MMO from M. capsulatus (Bath) compared to that found with enzyme from M. trichosporium OB3b.22 With the latter MMO the products were consistently found to have = 2:1 retention vs inversion of sterechemistry observed for both substrate enantiomers and for both ethane and butane substrates. Our work with the radical clock substrate probes implied a rebound rate constant of = 1013 s-1 for MMO from M. capsulatus

(Bath) and 3 - 6 1012 s-1 for MMO from M. trichosporium OB3b.2 5 It is possible that hydroxylation with the M. capsulatus (Bath) MMO system has more concerted than radical character. Alternatively, it may display a faster rebound rate compared to that in M. trichosporium OB3b system. Both explanations could account for the stereoselectivity of = 90 % retention found in some cases with the M. capsulatus (Bath) enzyme. Because some of the experiments with the M. trichosporium OB3b enzyme were carried out with identical batches of substrates reported here, it is unlikely that the different results are due to variations in the substrates or analytical procedures. Intramolecular kinetic isotope effects of = 4 with these substrates are consistently observed with MMO M. trichosporium OB3b.27 With MMO from M. capsulatus (Bath), low kH/kD values arise due to exchange. It is possible that an analogous reaction does not occur with the M. trichosporium OB3b enzyme. The intramolecular kH/kD values of = 4 agree with the figures obtained from the radical clock substrate work with M. capsulatus (Bath).25 In those experiments, an intramolecular isotope effect of = 5 was interpreted as indicating that substantial C-H bond breakage is involved in the 232 hydroxylation reaction step. With cytochrome P-450 hydroxylations, kH/kD values of 7 to 10 have been reported.15 The isotope effect is expected to be 2 1, the precise value depending on the linearity of the 0 ...H-C unit in the transition state.38 Alternative explanations for an inverse intramolecular kinetic isotope effect in the hydroxylation reaction are not obvious. The large C2/C1 regioselective effects observed here, ranging from 3 to 4, relative to intramolecular isotope effect literature values, = 1, are also explained by the exchange reaction and the access of the cacarbon to the hydroxylating species. Concluding Remarks. Although the foregoing interpretations are reasonably satisfying, some mysteries remain. The relatively large quantity of H 3HO that is produced cannot be explained by exchange alone. In some instances, this species is the largest peak detected in the 3H NMR spectrum of the reaction products. Some H3HO could be generated during substrate synthesis, since relatively large quantities of it have been detected in other work at the NTLF.39 The C2/C1 regioselective effects of = 3 to 4 were absent in work with the radical clock substrate probes. This large value suggests that the apparent regioselectivity of the reaction is not a good indicator of the intermolecular isotope effect. Instead, the substrate may have the ability to reorient itself preferentially in the active site prior to hydroxylation. Experiments to test the exchange reaction, by mass spectral analysis of the products with CD3CD 3 hydroxylation, and by repeating some of the reactions with the tritiated substrates are in progress by coworkers in our laboratory. The results of the work described in this chapter have raised some interesting questions about the hydroxylation mechanism. One very useful conclusion, however, is that MMOs from M. capsulatus (Bath) and M. trichosporium OB3b do exhibit different behavior, confirming variations seen 233 in experiments with radical clock substrate probes reported in Chapter 4 and redox titrations in Chapter 3. Determining the extent to which, and understanding why, the enzyme systems vary are topics for future work. 234 References (1) Anthony, C. The Biochemistry of Methylotrophs; Academic Press: New York, 1982, pp. 296-379. (2) Nguyen, H.-H. T.; Shiemke, A. K.; Jacobs, S. J.; Hales, B. J.; Lidstrom, M.

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Pilkington, S.; Papaefthymiou, G. C.; Dalton, H.; Hodgson, K. O.; Lippard, S. J. [. Am. Chem. Soc. 1991, 113, 9219-9235. 235 (15) Ortiz de Montellano, P. R. In Cytochrome P-450 Structure, Mechanism, and Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum Publishing Corp.: New York, 1986; pp 217-271. (16) McMurry, T. J.; Groves, J. T. In Cytochrome P-450 Structure, Mechanism, and Biochemistry; Ortiz de Montellano, P. R., Ed.; Plenum Publishing Corp.: New York, 1986; pp 1-28. ('17) Mansuy, D.; Battioni, P. In Activation and Functionalization of Alkanes; Hill, C. L., Eds.; Wiley: New York, 1989; chapter VI. (18) Guengerich, F. P. In Biological Oxidation Systems, Vol 1; Reddy, C. C.,

Hamilton, G. A. and Madyastha, K. M., Eds.; Academic Press: San Diego, 1990; pp 51-67.

(19) Ortiz de Montellano, P. R.; Stearns, R. A. J. Am. Chem. Soc. 1987, 109, 3415-3420.

(20) Bowry, V. W.; Ingold, K. U. J. Am. Chem. Soc. 1991, 113, 5699-5707.

(21) Bowry, V. W.; Lusztyk, J.; Ingold, K. U. J. Am. Chem. Soc. 1991, 113, 5687-5698.

(22) Priestley, N. D.; Floss, H. G.; Froland, W. A.; Lipscomb, J. D.; Williams, P. G.; Morimoto, H. J. Am. Chem. Soc. 1992, 114, 7561-7562. (23) Ruzicka, F.; Huang, D.-S.; Donnelly, M. I.; Frey, P. A. Biochem. 1990, 29,

1696-1700.

(24) Rataj, M. J.; Kauth, J. E.; Donnelly, M. I. J. Biol. Chem. 1991, 266, 18684-

1.8690.

(25) Liu, K. E.; Johnson, C. C.; Newcomb, M.; Lippard, S. J. J. Am. Chem. Soc. 1993, 115, 939-947. ('26) Pilkington, S. J.; Dalton, H. In Methods In Enzymology Academic Press: New York, 1990; Vol. 188; pp 181-190. ('27) Priestley, N.; Wilkinson, B.; Floss, H., unpublished results. 236 (28) Andres, H.; Morimoto, H.; Williams, P. G. J. Chem. Soc., Chem. Commun. 1990, 627-628. (29) Abbreviations: TMEDA = tetramethylethylenediamine; THF = tetra- hydrofuran; MOPS = N-morpholinopropane sulfonic acid; DMAP = 4- dimethylaminopyridine; DCCI = dicyclohexylcarbodiimide, SPA = specific activity. (30) Parker, I:). J. Chem. Soc. Perkin Trans. 2 1983, 83-88. (31) Andersson, K. K.; Elgren, T. E.; Que, L., Jr.; Lipscomb, J. D. J. Am. Chem.

Soc. 1992, 114, 8711-8713. (32) Newcomb, M., personal communication.

(33) Valentine, A. M.; Lippard, S. J., unpublished results.

(34) Choi, S.-Y.; Eaton, P. E.; Hollenberg, P. F.; Liu, K. E.; Lippard, S. J.;

Newcomb, M.; :Put, D. A.; Upadhyaya, S. P., manuscript in preparation. (35) Groves, J. T.; Viski, P. J. Org. Chem. 1990, 55, 3628-3634.

(36) Groves, J. T.; Viski, P. J. Am. Chem. Soc. 1989, 111, 8537-8538.

(37) Groves, J. T.; Stern, M. K. J. Am. Chem. Soc. 1987, 109, 3812-3814.

(38) O'Ferrall, R. A. M. J. Chem. Soc. (B) 1970, 785-790. (39) Morimoto, H.; Williams, P. G., personal communication. 237 Table 1. Component Amounts Used In Enzymatic Reactions at NTLF. Total reaction volumes were 1 ml. Protein B/ 25 mM MOPS, NADH (gl, Substrate Hydroxylasea Reductase pH 7 buffer 0.1 M stock) (mg) (gl) (l) (IS)-[1-2 H,1-3 H] - 13 (batch 1) 280 (batch 1) 500 50 ethane 13 (batch 1) 280 (batch 1) 500 50 ethane

(R)-[1-21,1-3H] - 13 (batch 1) 280 (batch 1) 500 100 ethane (S)-[1-2H,1-3H] - 15 (batch 2) 300 (batch 2) 400 100 ethane (R)-[1-2 H,1-3 H]-- 25 (batch 1) 560 (batch 1) 200 100 butane 25 (batch 1) 560 (batch 1) 200 100 butane (R-[2-3H]- 24 (batch 2) 450 (batch 2) 200 100 butane 24 (batch 2) 450 (batch 2) 200 100 butane 24 (batch 2) 450 (batch 2) 200 100 butane [2-3 H]-butane, 24 (batch 2) 450 (batch 2) 200 100 racemic mixture aSPA (propylene) = 200 mU/mg batch 1; 224 mU/mg batch 2 under usual conditions. SPA's of both batches under conditions to produce 1 to 2 mM product/ml was 40 mU/mg. Fe content ranged from 2.5 to 2.7 Fe/mol hydroxylase. 238

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CHAPTER 6.

Intermediates in, and Kinetic Studies

of the Reaction of Reduced Hydroxylase of

MMO from Methylococcus capsulatus (Bath) with Dioxygen 290 Introduction Methanotrophic bacteria play an important role in the balance of atmospheric methane and in the global carbon cycle.1 Methane is the sole source of carbon and energy for these organisms, which rely on the enzymatic system methane monooxygenase (MMO) to convert methane into methanol. The first step in the metabolic pathway is shown in equation (1).1 MMO exists in either in a particulate, membrane-bound form containing copper, or in a soluble form containing iron at the active site.2 Soluble MMO isolated from

+ CH4 + +H+ + NADH CH3OH + H20 + NAD (1)

Methylococcus capsulatus (Bath)3 or Methylosinus trichosporium OB3b,4 two of the better characterized systems, comprises three separate proteins, the hydroxylase, protein B, and the reductase. The hydroxylase component of MMO is a member of the non-heme carboxylate protein family which contain dinuclear iron units at their active sites. 5 Included are hemerythrin, the R2 subunit of ribonucleotide reductase, and purple acid phosphatase.6-8 We wish to understand the factors responsible for tuning the diiron center in each protein to perform its specific function, which ranges from the reversible binding of dioxygen in hemerythrin to its activation for the hydroxylation of methane in MMO. In pursuit of this objective, we are investigating the proteins of the MMO system and exploring the fundamental chemistry of the diiron center in the hydroxylase. The hydroxylase, the site for substrate binding as well as hydrocarbon activation reaction in MMO,4 contains up to two dinuclear iron centers and is isolated in its oxidized FeIIIFeIII (Hox) form. The mixed-valent FeIIFeIII 291 (Hmv),9 and fully reduced FelIFeII (Hred) redox states are readily accessible through chemical reduction of Hox. The crystal structure of Hox10 revealed that the Fe atoms are bridged by a hydroxide and a bidentate, semibridging glutamate ligand. Each metal is also coordinated to a histidine ligand. Two additional glutamate residues ligate one Fe atom in a monodentate fashion, whereas the second iron atom has a monodentate glutamate and a water molecule in its coordination sphere. In the sample studied by x-ray crystallography, the iron atoms are additionally bridged by an exogenous acetate ligand from the crystallization conditions. Other physical techniques, 11-14 such as EXAFS, EPR, ENDOR, and M6ssbauer spectroscopy, support this assignment for the coordination environment of the metal atoms. The reductase, containing one FAD and one 2Fe-2S cluster, accepts electrons from NADH and in turn reduces the dinuclear iron center in the hydroxylase. 1 5 4 7 Protein B with MMO from M. capsulatus (Bath) regulates electron transfer between the hydroxylase and the reductase. 18' 19 The effects of protein B in the M. trichosporium OB3b MMO system include its ability to lower the reduction potential of the hydroxylase,20 perturb the EPR signal of Hmv, 21 alter product distributions of the hydroxylation reaction,22 '2 3 and change the initial velocity of the completely reconstituted system.23 Little is known about the influence of the reductase and protein B on the reaction chemistry of the hydroxylase. Further examination of interactions among the three components, which influence their collective behavior in the MMO system, is necessary to elucidate fully the role of each protein in catalysis. Hred, the reactive species toward dioxygen, can be produced by chemical reduction of Hox with sodium dithionite in the presence of mediators. By using this approach, Hred was generated in the absence of protein B and 292 reductase, and its ability to hydroxylate hydrocarbons was explored. The substrate nitrobenzene produces highly colored products upon oxidation. Since neither Hred nor Hox have any optical absorption bands above 300 nm, this reaction was easily studied by optical methods. The effects of the reductase and protein B components on the kinetic constants, regioselectivity, and product yields of the reaction of Hred with nitrobenzene and dioxygen were determined. In the course of these studies, optical changes were detected in a control reaction of Hred with dioxygen even in the absence of substrate. Kinetic constants of these colored species were therefore determined as well, leading to their assignment as intermediates along the reaction pathway. 24 Parallel studies with MMO from M. trichosporium OB3b were reported while our work was in progress.25' 26 Spectroscopic and kinetic characterization of these intermediates was performed by using stopped-flow spectrophotometry and rapid freeze-quench EPR, Mbssbauer, and resonance Raman spectroscopy. We describe our findings in the above areas in the present article and suggest assignments for the intermediates in a proposed catalytic reaction cycle. Experimental Bacterial Growth and Protein Purification. Growth of the native M. capsulatus (Bath) organism and purification of the hydroxylase, protein B, and reductase components of its sMMO were performed as described elsewhere.14 27 Specific activities and iron contents were in the ranges reported.2 7 For Mdssbauer work, hydroxylase enriched with 57Fe was obtained from cells grown on usual medium 2 8 except that Na 5 7FeEDTA was added as the sole source of iron. Na 57 FeEDTA was prepared by dissolving 57Fe foil (52 mg, 0.91 mmole, 95% enriched, Cambridge

Isotope Labs) in 3.6 ml of 1 M ultrapure HNO 3. Next, 3.6 ml of H 20 and 0.58 g

(2 mmole) of Na4 EDTA.2H 20 were added. After stirring for 5 min, 30 mg 293 (0).75mmole) of NaOH was added to the iron solution. The resulting mixture was allowed to stir for 6 hours, after which time it was centrifuged to pellet the precipitate. The dark green supernatant was added to 100 ml 20 mM sodium phosphate buffer (pH 7.0). The resulting solution was autoclaved and added to the normal growth medium to a final iron concentration of 36 [tM. Specific activities with propylene and iron content of 57Fe enriched hydroxylase were in the reported ranges for native hydroxylase. Generation of Hred. To reduce the hydroxylase, a stoichiometric amount of a 10 mM solution of methyl viologen was added to Hox,, as an electron transfer mediator. The protein and mediator solution was then made anaerobic by evacuation and back-filling with argon for at least 5 cycles. Next, a 0.1 M solution of sodium dithionite was prepared by first degassing 2 ml of buffer (25 mM MOPS, pH 8.5) for 15 min with dioxygen-free argon. Degassed buffer was transferred with a gastight syringe to a vial which contained 41 mg (0.23 mmole) of sodium dithionite, previously made anaerobic by evacuation and back-filling with argon for 3 cycles. An aliquot of the sodium dithionite solution was then added to the protein solution by means of a gastight syringe to achieve a 2-fold molar excess concentration over that of the hydroxylase. Upon addition of the reductant, the reaction mixture immediately turned blue due to formation of the methyl viologen radical. Reduction was allowed to proceed for at least 45 min, during which time the reaction mixture was transferred to an anaerobic chamber (Vacuum Atmospheres). Inside this glovebox the protein solution was pipetted into dialysis tubing having a 10,000 molecular weight cutoff, and then dialyzed against 400 ml 25 mM MOPS, pH 7.0 buffer to remove methyl viologen and excess sodium 294 dithionite. After a 2 hour period, the protein was removed from the dialysis bag. Experiments with protein B and reductase were performed in two different ways. Reduction of Hox to Hred was first carried out, after which the other component(s) was added to the dialyzed protein. Alternatively, the additional component(s) was combined with Hox prior to reduction, with subsequent treatments as described. Single Turnover Reactions. Kinetic Studies. The reduced, dialyzed protein solution was diluted with 25 mM MOPS, pH 7.0 buffer to achieve a 50 gM concentration of Hred. Protein B concentrations ranged from 0 to 200 gM. The substrate nitrobenzene was added as the neat liquid, 10 gil per 1 ml of protein solution, to obtain saturation at approximately 5 mM. The resulting mixture was incubated for 5 min, after which time undissolved nitrobenzene was removed by pipette. For stopped-flow kinetic analysis, the protein mixture was loaded into a 10 ml Hamilton gastight syringe and then removed from the glovebox (vide infra). The reaction was initiated by mixing with dioxygen-saturated buffer, and optical changes at 404 nm were recorded to monitor the formation of 4-nitrophenol. Product Distribution and Yields. To determine the distribution of products from nitrobenzene hydroxylation, enzymatic reactions were typically performed with 8 mg (3.2 x 10-8 mole) of hydroxylase and variable quantities of protein B and reductase. Up to 2.2 mg (1.3 x 10-7 mole) of protein B and 5 mg of (1.3 x 10-7 mole) reductase were added to achieve ratios from 0 to 4 equivalents for a given component relative to the hydroxylase. The reaction mixture was reduced, dialyzed and incubated with substrate as outlined above. After dialysis, the mixture (=1 ml) was transferred to a 10 ml glass vial 295 which was then sealed with a septum. The vial was brought out of the box, and 0.5 ml of dioxygen-saturated buffer was injected by syringe through the septum. The resulting solution was placed in a shaking incubator maintained at 45 C and allowed to react for at least 15 min to insure completion of the reaction. To compare the regioselectivity of the catalytic system to that obtained in single turnover reactions, 1.5 mg (6 x 10-9 mole) of hydroxylase, 02 mg (1.2 x 10-8 mole) of protein B, 0.4 mg (1.2 x 10-8 mole) of reductase, and 1 gl (8 x 10-6 mole) of nitrobenzene were combined in a total volume of 0.5 ml. This mixture was incubated for 30 s at 45 C in a capped vial. By means of a syringe, 25 tglof a 0.1 M ethanol-free solution of NADH was then added through the septum and the mixture was allowed to react for a 15 min period at 45 °C. The distribution of 2-, 3-, and 4-nitrophenol products formed in enzymatic reactions with nitrobenzene was investigated through HPLC analysis. The reaction mixtures were treated with 200 l of a 3% trichloroacetic acid solution to precipitate the protein. The resulting suspension was centrifuged for 2 min in a table top centrifuge (Hill Scientific) to pellet the protein. An aliquot from the supernatant was then injected into the HPLC, and the identities of the reaction components were determined by comparison of their retention times with those of authentic standards of the expected products. Conditions for HPLC analysis were as follows. Stock solutions of 1 mM of 2-, 3-, and 4-nitrophenol and nitrobenzene were made in 25 mM MOPS buffer, pH 7.0 buffer. A 100 gl aliquot from each solution was injected into a Perkin-Elmer Series 4 high performance liquid chromatograph equipped with a reverse phase C18 radial compression column (-Bondapak, Waters). Detection was carried out at 300 nm, a wavelength at which all four 296 components had significant absorption. The solvent system used to elute the components was a methanol and water gradient with a flow rate of lml/min. The solvent composition was initially 60% water and 40% methanol, and was ramped to 100% methanol over a 20 min time period. The amount of each component present was calculated from the peak areas after normalization with the individual extinction coefficients at 300 nm. The extinction coefficients for the reaction components at 300 nm were determined in order to quantitate the HPLC peaks. Stock solutions, 10 mM in each phenol, were carefully diluted to approximately 5 x 10-5 M in 50 % buffer

(25 mM MOPS, pH 7.0) / 50 % CH30H. The UV/vis spectra were then recorded (Perkin-Elmer Lambda 7 spectrophotometer). The extinction coefficients were calculated from the absorbance at 300 nm at several concentrations and found to obey Beer's law. The relative yields of each product in the enzymatic reactions were then calculated from the HPLC traces by dividing the peak area by the extinction coefficient. The overall yields of products formed in a single turnover reaction of Hred with nitrobenzene were calculated by first determining the amount of 4- nitrophenol that was generated. The corresponding amounts of 2- and 3- nitrophenol for the appropriate ratio of protein B to hydroxylase as determined by the above HPLC analysis was next added to this value to determine the total amount of product formed. Finally, the amount of product was divided by the amount of dinuclear iron centers present in the reaction solution to determine the percent yield of the reaction. To optimize the yield of 4-nitrophenol in single turnover reactions, several conditions were investigated and their effects were compared. The incubation time to reduce Hox was varied from 30 min to 2 hours. Reactions with variable quantities of protein B were performed. Reductase alone and a 297 mixture of both protein B and reductase were added to the hydroxylase in parallel reactions. Nitrobenzene was also added prior to the addition of sodium dithionite to determine whether the order of addition of reductant and substrate affected the yield of this product. KM of Nitrobenzene. The KM constant for nitrobenzene in reactions with MMO was measured by monitoring the optical change at 404 nm. Reactions contained 3.3 mg (1.3 x 10-8 mole) of hydroxylase, 100 gl of B/reductase mix (= 2.6 x 10-8 mole in each protein), and 650 gl of a mixture of buffer (25 mM MOPS, pH 7.0) and a 10 mM stock nitrobenzene solution, the ratio of which depended on the amount of nitrobenzene desired in the reaction. A 50 gl portion of a 0.1 M NADH stock solution was added to initiate the reaction in a total volume of 1.2 ml. The mixture was then placed in a thermostatted cuvette at 45 C. The absorbance change at 404 nm over a 400 s time period was used to quantitate the amount of 4-nitrophenol produced. From the linear region of the change in product concentration vs time, the velocity of the reaction was determined. Velocities at several nitrobenzene concentrations were then plotted as a function of nitrobenzene concentration. From double reciprocal plots of these data, KM was cdetermined. Stopped-flow Spectrophotometry. Stopped-flow experiments were performed to monitor the reaction of in the presence of 60-200 gM protein B with dioxygen. This ratio of components was chosen because it gave optimal rate constants and yields from single turnover reactions with nitrobenzene (vide infra). Dialyzed solutions of Hred and protein B were loaded into a 10 ml Hamilton gastight syringe fitted with a KelF threaded adaptor for connection to a anaerobic line of a stopped-flow assembly (HiTech, Canterbury, England). The contents of 298 the 10 ml syringe were used to fill the drive syringe of the stopped-flow apparatus. By using a Matheson gas mixer, buffer (25 mM MOPS, pH 7.0) with variable concentrations of dioxygen (0 to 1 mM) was prepared, and then loaded into the second drive syringe of the stopped-flow assembly. Solutions were equilibrated for 30 min at a given temperature prior to mixing. To determine the pH dependence of the reaction of Hred with dioxygen, the anaerobic dialysis was performed with 400 ml of 10 mM MOPS, pH 7.0. The oxygenated buffer solution contained 100 mM MOPS buffer, with pH values ranging from 6.6 to 8.6. To test for the presence of a solvent deuterium isotope effect of the reaction velocities, hydroxylase was first exchanged into D)20 by using a procedure described elsewhere. 12 A small volume ( 175 tgl)of a 5 mM solution of protein B was added to the exchanged Hox (vol = = 2 ml) prior to reduction. A stock of 25 mM MOPS in D20 was used to make the 0.1 M sodium dithionite solution (pD = 8.5, uncorrected for the deuterium isotope effect) and for the dialysis and oxygenated buffer in the second drive syringe (pD = 7.0, uncorrected). For reactions with the substrates hexane and methane, the oxygenated buffer was further treated as follows. With hexane, 100 ml of dioxygen- saturated 25 mM MOPS, pH = 7.0 buffer was mixed with 50 ml of hexane for 20 min to obtain a saturated solution of this substrate. The aqueous layer was removed and used to fill the aerobic drive syringe. With methane, a similar procedure was used, except that 25 ml of dioxygen-saturated buffer was incubated with 25 ml of methane gas at approximately atmospheric pressure. Spectrophotometric analysis was performed with one of two detection systems. A photomultiplier tube was used to collect data at single wavelengths between 330 and 800 nm. Alternatively, a Hewlett Packard 299 UV/vis diode array spectrophotometer (model 8452A) was used to collect data at multiple wavelengths. Rapid Freeze-Quench Studies. M6ssbauer samples were made with 5 7Fe-enriched hydroxylase. Both freeze-quench EPR and M6ssbauer samples were prepared by using an Update Instruments rapid freeze-quench apparatus following methods outlined elsewhere. 2 9 Solutions of 600 - 700 jtM Hred and 1.2 - 1.4 mM protein B were mixed rapidly with a dioxygen-saturated ( 1 mM) 25 mM MOPS, pH 7.0 buffer solution at 4 C. Protein was allowed to react with dioxygen for fixed time periods (0.025 - 60 s) before being sprayed into isopentane at -140 C. A schematic diagram of the apparatus is illustrated in Figure 1. EPR Spectroscopy. EPR spectra were recorded at X-band on a Bruker Model ESP 300 spectrometer with an Oxford Instruments EPR 900 liquid He cryostat set at 9 K. The Hmv signal at gay = 1.83 was quantitated under non-saturating conditions by double integration of the first derivative spectrum for comparison to a frozen solution of copper perchlorate (lmM CuSO4, 2 M

NaC10 4, 0.01 M HCl). Transition probabilities were corrected for g-value anisotropy. 3 0 The percentage of Hmv species was calculated from the concentration of spin relative to the concentration of iron present in the sample. Mossbauer Spectroscopy. M6ssbauer spectra were collected by using either a weak-field M6ssbauer spectrometer equipped with a Janis 8DT variable-temperature cryostat, or a strong field spectrometer outfitted with a Janis 12 CNDT/SC SuperVaritemp cryostat encasing an 8 T superconducting magnet. A constant acceleration mode in a transmission geometry was used to operate both 300 spectrometers. The centroid of the room temperature spectrum of a metallic iron foil corresponds to the zero velocity of the Mbssbauer spectra. Resonance Raman Spectroscopy. Resonance Raman spectra of stopped-flow solutions were collected with excitation at several wavelengths (266, 350, and 406 nm). Protein concentrations as high as 150 JiM in Hred and 300 gM in protein B were employed these experiments. In addition, Raman spectra were collected on the freeze-quench EPR samples, made with either 1602 or 1802 (95% enriched, Cambridge Isotope Labs), by using Kr+ ion laser lines at 413 and 647 nm and a dewar and at assembly in the Spiro laboratory at Princeton University. Results Studies of Nitrobenzene Hydroxylation. Product Distribution and Yields in Single Turnover Experiments. HPLC traces of extracts from single turnover reactions of Hred and various ratios of protein B are illustrated in Figure 2. In all cases, the predominant product was 4-nitrophenol. By contrast, under catalytic conditions, in which all components were present and NADH sustains turnover, a mixture of 57% 4-nitrophenol and 43% 3-nitrophenol was produced. Product distributions under all conditions tested are summarized in Table 1. Optimal product yields were obtained with a 45 min incubation time of reductant with Hox and a subsequent 2 h dialysis period. In all cases where an additional component(s) was incubated with the hydroxylase, the results were the same irrespective of whether the protein(s) was added to either Hox or Hred. Yields from reactions of Hred with nitrobenzene in the presence of reductase were similar to yields observed with Hred alone. Incubation with both protein B and reductase resulted in low yields, approximately 75 % less than with the Hred alone. The addition of protein B had a dramatic effect on 301 the yield, tripling the absorbance change at 404 nm compared to a reaction with only Hred present. A plot of the percent yield of product versus the number of equivalents of protein B present in the reaction mixture is given in Figure 3. Greater amounts of 4-nitrophenol were detected at higher ratios of protein B relative to Hred. Addition of nitrobenzene prior to reduction of Ho,, had no effect on the yield of 4-nitrophenol. Kinetic Analysis of the Catalytic Hydroxylation of Nitrobenzene. Reactions with the catalytic MMO system and nitrobenzene were carried out to determine kcat and KM for this substrate. Traces of absorbance versus time for various substrate concentrations, a Michaelis-Menten plot, and the corresponding double reciprocal plot are presented in Figure 4. From these data, a value - 5 mM was calculated for KM. Kinetic traces from single turnover reactions of Hred and Hred plus 2 equivalents of protein B under pseudo-first order conditions in dioxygen and nitrobenzene are given in Figure 5. A rate constant was obtained from each trace after fitting to the first- order growth equation indicated in equation (2). A plot of kobs values obtained in this manner as a function of the ratio

y=A+B(1-e-kobs*t) (2)

of protein B to hydroxylase used in the reaction is given in Figure 6. From this plot it is apparent that the addition of protein B increases kobs from 0.011 s- 1 to a maximum of 0.40 s-1 with the addition of between 1.5 and 2.0 molar equivalents of protein B to hydroxylase. Further addition of protein B above this ratio causes kobs to decrease slightly. Above 2.5 equivalents the rate constant levels off to 0.25 s-1. Protein B therefore induces a maximal increase of - 33-fold in kobs. 302 Intermediates Formed in the Reaction of Hred with Dioxygen. Spectroscopic Characterization of Intermediates. Mdssbauer data were collected from samples quenched between 0.025 and 60 s after rapid mixing. Figures 7A, 8A, and 9A show representative spectra taken at three different time points. Over the time range investigated, five species in the form of quadrupole doublets were present at varying concentrations, M6ssbauer parameters of which are summarized in Table 2. The five species correspond to four different chemical forms of the dinuclear iron center, Hox, Hred, and two intermediates designated L and Q. The spectrum of Ho, shown in Figure 10A, has Mbssbauer parameters indicative of the oxidized, Fe(III)Fe(III) oxidation state of the diiron center ( = 0.51 + 0.02 mm/s and AEQ = 1.15 ± 0.03 mm/s for site 1, 6 = 0.49 ± 0.02 mm/s and AEQ = 0.85 + 0.03 mm/s for site 2). Two diferrous signals, referred to as Hred(l) and Hred(2), are attributed to Hred and both display parameters of 6 = 1.3 0.02 mm/s with AEQ = 2.8 + 0.03 mm/s. The weak and strong field Mdssbauer spectra of a mixture of these two signals are given in Figures 10B and 11A, respectively. In accord with our earlier report,2 4 the extracted M6ssbauer spectrum of intermediate L is a sharp symmetrical quadrupole doublet with parameters 6 = 0.66 + 0.02 mm/s and AEQ = 1.51 + 0.03 mm/s, as shown in Figure 7B. From subsequent stopped-flow optical spectrophotometric studies, carried out at single wavelengths by using fiber optics detection, an absorption band at = 625 nm with = 1000 M-1cm- 1 was estimated for L. Figure 12 shows the growth of this feature with time, measured at 625 nm, an analysis of which is given below. The optical spectrum of intermediate Q, shown for various time points in Figure 13, has Xmax values of 350 (£ = 3600 M-lcm-1l),420 (£ = 7200 M-1cm-1), and 520 nm ( = 14,000 M-1cm-1). Figure 9B shows the extracted M6ssbauer 303 spectrum of Q, which comprises two distinct doublets, having parameters = 0.21 + 0.02 mm/s and AEQ = 0.68 + 0.03 mm/s for doublet 1 and 6 = 0.14 ± 0.02 mm/s and AEQ = 0.55 + 0.03 mm/s for doublet 2. High field Mdssbauer spectra, shown in Figures 11B and 11C, reveal both intermediates L and Q to be diamagnetic. This observation agrees with low-temperature EPR spectral studies of L and Q presented in Figures 14 and 15, respectively. In these spectra, only signals corresponding to Hred (g = 15), Hmv (gav = 1.83, = 5% based on total Fe) and a small amount of a protein-associated free radical (g = 2.0, 5% based on total Fe), present in all oxidation states, were detected. Resonance Raman spectra of freeze-quench samples measured at reaction times of 10 ms, 155 ms, 8 s, and 60 s with excitation at 413 nm are given in Figure 16. Many peaks are apparent. Spectra for samples reacted with either 160, or 1802 appeared very similar. Excitation at 647 nm with the 155 ms sample revealed a peak at 902 cm- 1 which shifted by 23 cm-1 upon substitution, as summarized in Figure 17. These studies remain in progress. Kinetic Analysis. Kinetic analysis of the time dependent changes in the freeze-quench M6ssbauer signal revealed several important aspects of the reaction of Hredi with dioxygen. The reaction proceeds as given in equation (3), and plots quantitating the variation in concentration of each of these species over time are given in Figure 18. The data were fit to equation (4), which describes a system containing three consecutive first order processes.

k, k2 k3 Hre d L Q ~ H(3)

klk2k3 e-kl*t e-k2*t e-k3*t 1 1 1 Y=I k2-kl Lkl(k3-kl) k2(k3-k2) k3 k3-k2 k3-kl 304

-1 The rate constant k1, = 25 s , corresponds both to the decay of Hred and to the growth of intermediate L. The decay of L occurs with a rate constant of k2 0.3 s- 1, which matches the rate constant extracted for the growth of - intermediate Q. Hox then forms with a rate constant k3 0.03 s 1, corresponding to the rate constant for the decay of Q. A significant amount of Hred remained after 30 s, and M6ssbauer analysis revealed that this species is converted to Hox with a much smaller first order rate constant. The contributions of the species to the Md6ssbauer spectra were subtracted during the above analysis (vide infra). Stopped-flow spectrophotometry was also used to analyze the kinetics of formation and decay of both intermediates L and Q. For L, detection at 625 nm resulted in the kinetic trace shown in Figure 12. These data were fit to a first order growth equation, giving k1 22 sl. Studies of Q at 420 nm resulted in the kinetic trace shown in Figure 19A. Trace A was fit to the expression given in equation (5) which contains two consecutive processes, with

- = - corresponding rate constants of k2 = 0.5 + 0.15 s 1 and k3 0.07 + 0.02 s 1, respectively. Trace B in Figure 19 was obtained under identical conditions except that protein B was omitted.

k2*A y = k2 [B*(1 - e- k2*t)+ C*e-k3*t] (5) k2- k3

The kinetic constants k2 and k3 measured by stopped-flow spectrophotometry under a variety of conditions are listed in Table 3. These rate constants were unaffected by the dioxygen concentration, as shown in Figure 20. Similarly, the rate constants were pH independent in the range 6.6

<; pH 8.6, as indicated in Figure 21. When the reaction was run in D20, 305 there was little effect on the rate constants. A kinetic trace for the reaction run under these conditions is illustrated in Figure 22, and fitting to the

- - biexponential equation yielded rate constants of k2 = 0.35 s 1 and k3 = 0.15 s 1 at 6 °C. The temperature dependence of the rate constants for the formation and decay of compound Q was also measured. Arrhenius and Eyring plots of these data are given in Figures 23A and 23B, respectively. The parameters determined from these plots are listed in Table 4. The optical spectrum of Q was also recorded in the presence of substrate. With methane, high protein concentrations (> 150 M) were necessary to detect this intermediate. Fits the observed kinetic traces, an example of which is shown in Figure 24, revealed both k2 and k3 to increase slightly, to values of = 0.95 s-1 and 0.17 s-1, respectively. Analogous data were collected with the substrate hexane. The corresponding kinetic trace was fit = - with k3 0.13 s 1, but k2 was unchanged. The optical spectrum of Q, taken in the presence of' either methane or hexane, revealed no new chromophoric species present. Data with nitrobenzene differ from those collected with methane and hexane since the product of the hydroxylation reaction, 4- nitrophenol, absorbs significantly at 400 nm. When monitored at 420 nm, k2 -1 - . was determined to be 0.26 s and, k3 0.11 s 1 No other new optical bands were detected. Discussion MMO Component Interactions as Assessed by Hydroxylation of Nitro- benzene. The interactions of protein B and reductase with the hydroxylase were evaluated through the influence of these two components on the hydroxylation of nitrobenzene by Hred. Three aspects of this reaction were investigated, the regioselectivity of hydroxylation, the overall product yield, 306 and the rate constant for product formation. Examination of the product alcohols indicated that hydroxylation of nitrobenzene proceeds in a regioselective manner depending on the reaction conditions. With chemically reduced hydroxylase (Hred), 4-nitrophenol is the predominant product irrespective of the amount of added ratios of protein B up to a fourfold excess. Under catalytic conditions with all components (Hox, protein B, reductase, N'ADH) present, the distribution of products changed to an approximately equimolar mixture of 4-nitrophenol and 3-nitrophenol. The presence of reductase and NADH, therefore, alters the regioselectivity of hydroxylation to afford 3-nitrophenol, which was not produced by protein B alone. These findings are compared in Table 1 to the results of analogous studies of catalytic reactions with MMO from M. trichosporium OB3b.2 3 In this MMO system, unlike MMO from M. capsulatus (Bath), protein B is apparently not required to sustain turnover under catalytic conditions,4 and H202 can support high levels of turnover in a shunt pathway with Hox alone.2 2' 2 3 31 Investigation of the regioselectivity of nitrobenzene hydroxylation with the M. trichosporium OB3b system2 3 revealed that 2 90% of the product is 3-nitrophenol under catalytic conditions without protein B. Adding 0.1 to 2 equivalents of protein B to hydroxylase, reductase, and NADH afforded 83 to 89% of 4-nitrophenol. In this case, therefore, protein B shifts the product distribution toward 4-nitrophenol. In the H20 2 shunt pathway of Hox from M. trichosporium OB3b,22 almost equal amounts of 4-nitrophenol and 3-nitrophenol were produced, which matches behavior seen with the catalytic system from M. capsulatus (Bath). It is also noteworthy that the KM (-::5 mM) for nitrobenzene with the catalytic MMO system from M. capsulatus

(Bath) matches the KM value of Hox and H2 0 2 , but not the KM value ( 100 307 pM) for the catalytic system from M. trichosporium OB3b. From these results and the data in Table 1 it is obvious that the component interactions in the two systems are complex and alter the hydroxylation chemistry in different manners. Significant formation of 4-nitrophenol is somewhat surprising if the hydroxylation mechanism involves electrophilic attack on the aromatic ring. The nitro substituent is a powerful deactivating group and a strong meta director, and if substrate reactivity were the determining factor, 3-nitrophenol might be expected to be the major product. We therefore suggest that the stereochemistry of the substrate binding in the active site determines the regioselectivity of the reaction. This property will be affected by the presence of the other protein components, since their interactions may cause perturbations in the active site structure.3 2 An alternative possibility is that the hydroxylation mechanism does not involve electrophilic aromatic substitution. The product yield in single turnover reactions with Hred is also affected by the presence of the other components. Since HPLC analysis identified 4- nitrophenol as the predominant product under all conditions with Hred, the increased yields are not due to a shift in product distribution. Protein B markedly increased the yields, as illustrated in Figure 3. Addition of protein B and reductase to Hred resulted in diminished yields, and when added to Hox prior to reduction, extremely low levels of hydroxylation were observed. The latter finding is consistent with the difficulty reducing Hox to Hred with protein B and reductase present, as monitored by EPR spectroscopy.l9 Adding reductase alone to Hox or Hred had very little effect on the yields relative to reactions with Hred alone. These results imply that protein B, and not the reductase, increases the yield of the hydroxylation reaction of nitrobenzene. 308 It is possible that reduction of the complex formed by Hox,,with protein B and reductase might produce Hred that would be a good hydroxylation agent when dioxygen is added. This alternative is consistent with increased reduction potentials of the substrate/reductase/hydroxylase complex relative to Hox alone. Such a reactive Hred species formed in this manner cannot be investigated under single turnover conditions, however, since reduction of Hox to Hred is inhibited by protein B and reductase in the absence of substrate, and nitrobenzene, the substrate in these experiments, prevented the mediated reduction of Hox to Hred. Addition of protein B and reductase to Hred may not produce the same reactive species because of likely structural alterations produced along with the change in oxidation state of the hydroxylase. Protein B also increases the rate constant for single turnover reactions of Hred with nitrobenzene. Neither addition of reductase to Hox or Hred, nor of both protein B and reductase to Hred, increased the rate constant relative to reactions of Hred alone. These observations again imply that protein B alone activates the hydroxylase and that protein B and reductase do not significantly affect Hred. The pseudo-first order rate constant for hydroxylation increased by up to 33-fold when Hred was treated with protein B. This behavior is reminiscent of that reported for protein B from M. trichosporium OB3b for titrations performed with Hox,,and reductase under catalytic turnover conditions. 21 In contrast to the present single turnover reactions of nitrobenzene with Hred from M. capsulatus (Bath), protein B had little effect on the rates of analogous reactions of Hred from M. trichosporium OB3b.23 Instead, protein B from M. trichosporium OB3b increased the initial velocity of the complete reconstituted system (Ho, reductase, NADH, catalytic conditions), but decreased initial velocities in reactions of Hox with H20 2 . As seen in the product distribution studies, component interactions affect the 309 kinetic behavior in a complex manner which differs for the two MMO systems. Complex formation among the three protein components of M. trichosporium OB3b MMO has been demonstrated by use of chemical cross- linking agents and other methods.21 In this study, it was hypothesized that protein B bind s tightly to Hox to form an activated complex, but then dissociates from Hred after electron transfer occurs. The present results are consistent with initial binding of protein B to Hox to form an activated complex. Because identical behavior is seen when protein B is added to Hred, we conclude that protein B can bind to Hred to form an activated complex as well. If the Hred-protein B complex is the species responsible for the increased rate constants, and Hred and protein B are in equilibrium with one another, then the addition of more protein B would ensure that Hred is in the protein B-bound complex. Elevated concentrations of protein B beyond two equivalents per Hred may diminish the rate constant of the reaction by sterically hindering substrate binding to Hred. The total yield of the reaction could still remain high, since nearly all of the Hred is in the active, protein B- bound form. If there are adventitious binding sites for additional molecules of protein B beyond two equivalents, excess protein B would have little effect once all such sites were fully occupied. This model is consistent with the B dependence of kobs shown in Figure 6 and with the yields reported in Figure 3. Close examination of Figure 3 indicates that some cooperative behavior may be taking place. It is possible that binding of protein B to one dinuclear iron site in Hred increases the binding affinity for the second site. The errors of these data points are large, however, which may account for the apparent curvature in the data. 310 The M. capsulatus (Bath) reductase alters the product distributions of Hred with nitrobenzene, but protein B plays many more roles in the MMO system. As mentioned earlier, it regulates electron transfer from the reductase to the hydroxylase so as to occur only in the presence of substrate; 17 it shifts the reduction potentials of the substrate/reductase/hydroxylase complex; 19 and it affects kobs in single turnover reactions of Hred. Protein B from M. trichosporium OB3b alters the reduction potential of Hox,20 perturbs the EPR signal of Hmv,21 changes product distributions with Hred,23 and affects the initial velocity of the complete reconstituted system and the H202 shunt system. 23 A molecular explanation for these observed functions remains a challenge for the future. More structural work on component interactions with both MMO systems is essential to understand fully the fundamental reasons for the observed effects on the rate constants and yields of the reactions.

Spectroscopic Studies of Intermediates in the Reaction Cycle. Because optimal yields and rate constants under single turnover conditions occurred in the presence of 2 equivalents of protein B, these conditions were adopted for investigating the reaction of Hred with dioxygen. Reactions were carried out in the absence of substrate in order to allow detectable concentrations of intermediates to accumulate, since these activated species were presumed to react quickly with substrate. The temperature was also lowered to 4 C to slow down the reaction and facilitate the detection of short-lived species. In earlier communications we reported the optical spectroscopic and M6ssbauer parameters of intermediates L and Q and the resonance Raman features of L formed in the reaction of Hred with MMO from M. capsulatus (Bath) with dioxygen.2 4 33 For intermediate L, a doublet with parameters of 6 311 = 0.66 + 0.02 mm/s and AEQ = 1.51 + 0.03 mm/s was observed. Carboxylate- bridged high spin diiron(III) complexes generally have isomer shift values between 0.45 and 0.55 mm/s,6 -8 and isomer shift values for diiron(II) compounds fall in the range of 1.1 - 1.3 mm/s. 6 -8 Although parameters matching the values of L have not been observed in model compounds, the magnitude of the isomer shift is close to parameters seen for diiron(III) clusters. Since Hred decays with concomitant production of intermediate L, the minimal mechanism dictates that L is an early species formed in the reaction of Hred with dioxygen. Diiron(III) peroxide complexes, suggested as products in reactions of several diiron(II) model complexes with dioxygen, result from two-electron transfer from the Fe atoms to the dioxygen ligand.5 Based on the kinetic data, Raman spectra (vide infra), and M6ssbauer parameters, we propose L to be a diiron(III) peroxide intermediate in the catalytic reaction cycle of MMO. Isomer shift values of 6 = 0.52 - 0.54 mm/s have been reported for a few diiron(III) peroxide complexes.34-36 In general, isomer shift values increase according to increasing coordination number and negative charge on the complex. 37 The high 6 value of 0.66 mm/s for L could indicate the presence of six-coordinate iron atoms and considerable peroxide-to-iron charge transfer character. A coordination number of seven for one or both of the iron atoms could also account for the high isomer shift. Among other possibilities, an ii1,T1 peroxide with iron coordination numbers of six, or an a T2,T2 binding mode with seven-coordinate iron atoms are both consistent with this proposal. The sharp, symmetrical shape of the M6ssbauer signal strongly implies that the iron atoms are in nearly identical coordination environments. The optical band at 625 nm ( = 1000 Ml1cml) for L is close to 312 that reported (ma, = 600-610 nm) for several diiron(III) peroxide complexes. 3 4 ,3 8 ,39 The Raman spectrum,3 3 obtained at an excitation wavelength of 647 nm, of a rapid freeze-quench sample, frozen 155 ms after mixing to optimize the quantity of L formed, exhibits a feature at 902 cm- 1 which shifts with 180 substitution. This frequency is in the reported range for u(O-O) of several iron(III) peroxide model complexes, and provides strong evidence for the existence of such a unit in L. The 902 cm-1 feature was much less enhanced in spectra obtained with excitation at 413 nm, implying that the 625 nm optical absorption is a peroxide-to-iron(III) charge transfer band. The Au of 23 cm -1 observed with the use of 1802 is less than the 42 cm-1 shift predicted for a diatomic harmonic oscillator. Coupling of the 0-0 stretching mode to another mode in the diiron unit may lead to a lower value for Atu. A second possible explanation is that the bound peroxide exchanges with the oxygen atom of the bridging hydroxide ligand, since the Au value matches that expected for 160-180 substitution. A vibration characteristic of v(Fe-0 2) between 400 and 500 cm-1 was not observed. The geometry of excited state strongly affects the intensity of this stretch, which was not observed in several peroxide complexes, including including oxyhemacyanin.4041 Figure 25A proposes structures for L that are compatible with the spectroscopic data. The optical and Raman spectroscopic properties of L rule out other models proposed previously. 2 4 For example, a delocalized mixed valent system with a coordinated superoxide anion radical arising from single electron transfer from the iron(II) atoms was suggested to account for the high 6 value. Another alternative was that L might be a dioxygen adduct of a diiron(II) unit which is antiferromagnetically coupled with intermediate spin

(S = 1). 313 The decay of L proceeds with concomitant formation of Q. This second intermediate has been observed in both the M. capsulatus (Bath)24 and M. trichosporiunm OB3b MMO systems. 2 6 The M6ssbauer spectrum of Q from M. trichosporium OB3b is a doublet with 6 = 0.17 mm/s and AEQ = 0.53 mm/s. Since this doublet appeared to be symmetrical, the iron atoms were assumed to reside in equivalent coordination environments. In the M. capsulatus (Bath) system, two unresolved doublets of equal intensity were readily distinguished and fit to the parameters given in Table 2. The average isomer shift and quadrupole splitting of the two doublets, 0.18 mm/s and 0.62 mm/s, respectively, agree with parameters obtained from the corresponding spectrum of Q from the M. trichosporium OB3b organism. In the M. capsulatus (Bath) system, however, the presence of two distinct signals for Q indicated that the iron atoms have in inequivalent coordination spheres. The average isomer shift value of 0.18 mm/s is outside the range for carboxylate-bridged, octahedral high-spin diiron(III) clusters.6' 7 Instead, the value is near the range reported for the few characterized heme and non- heme iron(IV) complexes,4 2 4 5 suggesting that Q might be an iron(IV) oxo species. Figure 25B illustrates such possible structures for Q. The inequivalence of the Fe sites could reflect significant rearrangement of the protein ligands in Q as compared to L and Hox. Alternatively, the oxygen atom of the proposed Fe(IV) oxo species may not be bound symmetrically between the Fe atoms. A species having a low spin diiron(III) unit with a coordinated oxyl ligand, which is antiferromagnetically coupled to a cysteinyl radical, has also been suggested for Q.5 The widely accepted hydroxylation mechanism for the heme protein cytochrome P-450 similarly invokes an Fe(IV) oxo species which abstracts a hydrogen atom from the substrate molecule.4 6 Cytochrome P-450 does not 314 hydroxylate methane, however. Very recently, Fe(IV) intermediates have been ruled out in the mechanism for the generation of the tyrosyl radical in the R2 protein of ribonucleotide reductase, which has an active site structure very similar to that of the MMO hydroxylase.2 9 4 7 48 Instead, a diiron(III) oxygen radical species was implicated in the oxidation of the tyrosine residue. A similar species, perhaps the oxyl radical suggested above, which is reactive toward CH4 may be present in MMO. The time course freeze-quench M6ssbauer study revealed that two types of iron centers exist in Hred. Of the = 0.7 mM concentration Hred used in this study, only = 45% was consumed after 8 s, at which time L had been converted to Q. Lower Hred concentrations, 420 M, resulted in approximately the same percent consumption at 8 s, thus ruling out the possibility that dioxygen was a limiting reagent at the higher protein concentrations. Close examination of the M6ssbauer spectrum of Hred in Figure 10B reveals the existence of two types of iron signals, designated Hred(1) and Hred(2). The high-field Mdssbauer spectrum in Figure 11A confirms this finding. The time dependence of one of these signals, attributed to Hred(2 ), revealed that it reacts with dioxygen with a slower rate constant of

= 0.1 s-1. The EPR spectrum of a freeze-quench sample made after 8 s of reaction time shows a distinguishable signal at g = 15, further confirming the persistence of Hred(2), although accurate quantitation of this resonance was not possible. M6ssbauer analysis of Hred revealed that Hred(l) and Hred(2) are present in roughly equal amounts in the sample. Both forms of Hred react with dioxygen. Hred(l) reacts rapidly and produces L, while Hred(2 ) reacts more slowly to produce Hox. The latter reaction does not produce any distinguishable intermediates, however. This finding is consistent with the fact that maximal yields of only = 40% are observed in single turnover 315 reactions of nitrobenzene and Hred and dioxygen. Presumably, conversion results only from Hred(l). Similar heterogeneity in Hred from M. trichosporium OB3b may not be as pronounced, since the g = 15 signal of Hred was reported to disappear completely with a rate constant of = 22 s-1.26 On the other hand, single turnover reactions with this system do not fully convert nitrobenzene either.

The extinction coefficents previously reported for Q,2 4 £ = 1800 M-lcm -1 (350 nm), = 3600 M-lcm -1 (420 nm), and e = 7000 M-lcm -1 (520 nm) are adjusted here to values of 3600, 7200, and 1400 M-lcm-1 , respectively, to reflect the presence of the inactive Hred clusters. For L, this adjustment was already considered in the calculation of £ : 1000 M-lcm-1 at 625 nm. Stopped-flow kinetic studies of Q with optical detection at 420 nm yielded rate constants for the growth and decay of this intermediate that were fit cleanly to a biexponential equation with a single growth and a single decay phase. These rate constants matched within error those determined from the kinetic M6ssbauer data (Table 3). In studies with M. trichosporium OB3b, the growth and decay of Q were best fit to a triexponential equation, which included two separate growth curve components. 26 The origin of these separate processes was attributed to discrete Hred/B complexes that react with cdioxygen with different rate constants to form Q, behavior which was not observed in the M. capsulatus (Bath) system. The average rate constant for growth, 1 0.1 s-l1,was about twice the value reported here for M. capsulatus (Bath), but both MMO systems had similar decay constants of = 0.05 to 0.065 s-1. The optical spectrum of an unexplained species that formed but did not further react was observed in the reaction with M. trichosporium OB3b, and was subtracted from the observed spectrum to reveal that of Q.26 This species may correspond to Hred(2) observed with M. capsulatus (Bath), but may be 316 present in smaller quantities, rendering its EPR signal undetectable. Alternatively, it could be EPR silent in this system. Kinetic traces for the formation and decay of Q were recorded under a variety of conditions. Without protein B, very little Q was detected, as shown in Figure 18B. Addition of protein B to Hred yielded the kinetic trace in Figure 18A. These observations are consistent with behavior seen with reactions carried out under single turnover conditions of Hred with nitrobenzene, and indicate that protein B activates Hred. Conversion of L, a diiron(III) peroxide complex, into any of the two species proposed above for Q requires cleavage of the 0-0 bond followed by the release of a water molecule. From the temperature dependence of the rate constant for this process, a large ASt value of 147 + 5 Jmole-1 K- 1 was calculated, which is consistent with release of a water molecule. The corresponding ASt value of = 8 + 0.5 Jmole- 1 K-l1 for the decay of Q is low, as expected for reaction of Q with substrate, to yield Hox and the alcohol product. The significant AHt values, = 113 ± 4 kJmole -1 (Ea = 113 ± 4 kJmole - 1) for the formation and = 75 3 kJmole- 1 (Ea = 75 + 3 kJmole -1) for the decay, are consistent with significant bond rearrangement occurring in these steps. Rate constants for both the formation and decay of Q were independent of the concentration of dioxygen. This observation is consistent with L being the intermediate formed from the reaction of Hred with dioxygen. The rate constant for L formation is expected to have a first-order dependence on the concentration of dioxygen in the system. Study of L by stopped-flow techniques has proved to be more difficult, owing to its lower extinction coefficient and shorter lifetime and remain in progress. Because L formation is most likely irreversible and proceeds with a rate constant ( 25 s- 1) which is nearly two orders of magnitude greater than the rate constant for Q formation 317 (- 0.4 s-l), the latter should only be dioxygen-dependent at very low 02 concentrations, which are experimentally impractical to attain. Lack of dioxygen dependence as measured through intermediate Q is therefore consistent with a mechanism in which Hred reacts with 02 to produce L which then decays to form Q. Since the reaction catalyzed by the MMO system requires two protons, the effects of pH and a D20 medium on the kinetic behavior of Q were - investigated. Except for an increase in k3 with D2 0 to values 0.145 s 1, no differences were observed. This finding does not address whether or not proton transfer occurs in either of these steps, but rather indicates that proton transfer is not rate determining. If proton transfer were rate determining in either the conversion of L to Q or in the decay of Q, a significant decrease in k2 or k3 would be expected. Another process, such as cleavage of the 0-0 bond, may be rate determining in Q formation. The presence of the substrates methane, hexane, and nitrobenzene caused much less Q to accumulate when compared to reactions run in the absence of a hydrocarbon substrate. With methane, Q was detected only at h:igh concentrations of Hred, and both k2 and k3 increased. With both hexane and nitrobenzene, k2 was not affected significantly, as shown in Table 3, which implies that these substrates had little effect on the conversion of L to

Q. Since k3 increased and much less of intermediate Q was detected in all cases, the substrates are assumed to react with Q. In no case were additional optical bands were detected following the decay of Q. This result contrasts with hydroxylation of nitrobenzene in the M. trichosporium OB3b system where an intermediate, termed T, was detected. 2 6 Intermediate T was observed only with nitrobenzene, and was attributed to an optical band a:rising from the bound product alcohol, the majority of the absorbance at 430 318 nm arising from 4-nitrophenol. This difference may reflect the different binding affinities of the product to the active site in the two systems. Conclusions. Single turnover reactions of Hred with nitrobenzene are highly dependent on the concentration of protein B in the system. Both the yield and the rate constant of the reaction are optimized with two equivalents present. The regioselectivity of the reaction under these conditions differs substantially from reactions performed under catalytic conditions with all components present, where the reductase plays a key role. By lowering the temperature and omitting substrate from the above reactions, two intermediates, L and Q, along the reaction pathway were identified and kinetically characterized. Structures consistent with the Mbssbauer, EPR, and optical spectroscopic characterization of these intermediates were proposed and related to one another in a catalytic cycle. Unusual M6ssbauer parameters for L and Q were observed, which will require the synthesis of appropriate model complexes for definitive structural characterization. From the foregoing findings, we propose the catalytic hydroxylation cycle for MMO from M. capsulatus (Bath) illustrated in Figure 26. The reaction begins with the resting state of the enzyme, Ho. Binding of substrate and subsequent electron transfer through the reductase yields the Hred- substrate complex. Dioxygen next binds to Hred and forms intermediate L, a diiron(III) peroxide. Rearrangement of L occurs with release of a water molecule and to produce intermediate Q, of uncertain composition and structure, which then reacts with substrate. Product dissociation leaves Hox, and the cycle is ready to begin again. Rate constants for each step are as indicated in the figure, and activation parameters are consistent with this proposal. 319 Acknowledgments. Rapid freeze-quench samples were prepared with the assistance of Professors B. H. Huynh and D. E. Edmondson at Emory University. Mssbauer samples were analyzed by Dr. D. Wang and Prof. Hluynh. Resonance Raman data were collected by D. Qiu and Professor T. G. Spiro. I thank A. M. Valentine for experimental assistance and helpful discussions, and Dr. A. Salifogolou for providing cell paste. I am also grateful to Dr. Y. Wang for driving to Princeton to initiate the Raman collaboration, even though I slept almost the entire way! 320 References (1) Anthony, C. The Biochemistry of Methylotrophs; Academic Press: New York, 1982, p 296-379.

(2) Scott, D.; Brannan, J.; Higgins, I. J. J. Gen. Microbiol. 1981, 125, 63-72. (3) Colby, J.; Dalton, H. Biochem. J. 1978, 171, 461-468. (4) Fox, B. G.; Lipscomb, J. D. Biochem. Biophys. Res. Comm. 1988, 154, 165-

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(8) Wilkins, R. G. Chem. Soc. Rev. 1992, 171-178. (9) Woodland, M. P.; Patil, D. S.; Cammack, R.; Dalton, H. Biochim. Biophys. Acta 1986, 873, 237-242.

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AW.A.; Lipscomb, J. D.; Minck, E. J. Am. Chem. Soc. 1993, 115, 3688-3701.

(12) DeRose, V. J.; Liu, K. E.; Kurtz, D. M., Jr.; Hoffman, B. M.; Lippard, S. J. J.

Am. Chem. Soc. 1993, 115, 6440-6441.

(13) Hendrich, M. P.; Fox, B. G.; Andersson, K. K.; Debrunner, P. G.; Lipscomb, J. D. . Biol. Chem. 1992, 267, 261-269.

(14) DeWitt, J. G.; Bentsen, J. G.; Rosenzweig, A. C.; Hedman, B.; Green, J.; Pilkington, S.; Papaefthymiou, G. C.; Dalton, H.; Hodgson, K. O.; Lippard, S. J. J. Am. Chem. Soc. 1991, 113, 9219-9235. (15) Lund, J.; Dalton, H. Eur. J. Biochemistry 1985, 147, 291-296. 321 (1.6) Lund, J.; Woodland, M. P.; Dalton, H. Eur. J. Biochem. 1985, 147, 297-

305.

(1.7) Green, J.; Dalton, H. Biochem. J. 1989, 259, 167-172. (1.8) Green, J.; Dalton, H. J. Biol. Chem. 1985, 260, 15795-15801. (19) Liu, K. E.; Lippard, S. J. J. Biol. Chem. 1991, 266, 12836-12839.

(20) Paulsen, K. E.; Liu, Y.; Fox, B. G.; Lipscomb, J. D.; Miinck, E.; Stankovich, M. T. Biochemistry 1994, 33, 713-722.

(21) Fox, B. G.; Liu, Y.; Dege, J. E.; Lipscomb, J. D. J. Biol. Chem. 1991, 266, 540-550.

(22) Andersson, K. K.; Froland, W. A.; Lee, S.-K.; Lipscomb, J. D. New J. Chem. 1991, 15, 411-415. (23) Froland, W. A.; Andersson, K. K.; Lee, S.-K.; Liu, Y.; Lipscomb, J. D. J. Biol. Chem. 1992, 267, 17588-17597.

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Lippard, S. J. J. Am. Chem. Soc. 1994, 116, 7465-7466.

(25) Lee, S.-K.; Fox, B. G.; Froland, W. A.; Lipscomb, J. D.; Miinck, E. J. Am. Chem. Soc. 1993, 115, 6450-6451. (26) Lee, S.-K.; Nesheim, J. C.; Lipscomb, J. D. J. Biol. Chem. 1993, 268, 21569- 21577.

(27) Liu, K. E.; Johnson, C. C.; Newcomb, M.; Lippard, S. J. J. Am. Chem. Soc. 1992, 115, 939-947. (28) Pilkington, S. J.; Dalton, H. In Methods In Enzymology Academic Press: New York, 1990; Vol. 188; pp 181-190.

(29) Ravi, N.; Bollinger, J. M., Jr.; Huynh, B. H.; Edmondson, D.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8007-8014. (30) Asa, R.; Vnngard, T. J. Mag. Res. 1975, 19, 308-315. 322 (31) Froland, W. A.; Andersson, K. K.; Lee, S.-K.; Liu, Y.; Lipscomb, J. D. In IUCCP Symposium on Applications of Enzyme Biotechnology; Plenum Press:

College Station, TX, 1991; pp 39-54. (32) See discussions in refs. 9 and 31. (33) Liu, K. E.; Valentine, A. M.; Qiu, D.; Edmondson, D. E.; Huynh, B. H.;

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1.990, 112, 5637-5639. 323 (4.4) Kostka, K. L.; Fox, B. G.; Hendrich, M. P.; Collins, T. J.; Rickard, C. E. F.;

Wright, L. J.; Miinck, E. J. Am. Chem. Soc. 1993, 115, 6746-6757. (45) Vogel, E.; Will, S.; Tilling, A. S.; Neumann, L.; Lex, J.; Bill, E.; Trautwein, A. X.; Wieghardt, K. Angew. Chem., Int. Ed. Engl. 1994, 33, 731-735. (46) Ortiz de Montellano, P. R. In Cytochrome P-450 Structure, Mechanism, and Biochemistry; Ortiz de Montellano, P. R., Eds.; Plenum Publishing Corp.: New York, 1986; pp 217-271. (47) Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8015-8023.

(48) Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8024-8032. 324

0

I QN ON ,-

I-4.- C)r. zo I-75 A V 4-

C) 0o N 0 C I o O ) - n 00 Ln 0 -4.

crall a

U,u 0. 0o >c, o a U r~ :t: 0) 0) 4-k

rr o0) 0 z -40 C) C

o4- cU ~C) E X. x >< x -z OC) cEo o 0 co o o +1 C)~ C)o© m( a o.E o O ~~63:~C C

-4 Ur4 75 O .C=j~ C) k C~l ct: CU cO fEC cra 325

Table 2. M6ssbauer Parameters of Species Detected in Rapid Freeze-Quench Samples from the Reaction of Hred with Dioxygen. Species 8 + 0.02 mm/s AEQ ± 0.03 mm/s

Ho, site 1 0.51 1.15

Ho, site 2 0.49 0.85

Hred(1) 1.3 2.8

Hred(2) 1.3 2.8

L 0.66 1.51

Q (site 1) 0.21 0.68 .~ Q (st(site 2))0.405 0.14 0.55 326

Table 3. Rate Constants for Formation (k2) and Decay (k3 ) of Intermediate Q Under a Variety of Conditions. Unless indicated otherwise, experiments were carried out at 4 °C. - Experiment k2 (s 1) k 3 (s-1) Mbssbauer Time Course 0.3 + 0.15 0.03 + 0.02

Stopped-Flow 0.5 + 0.15 0.07 + 0.02

Varying [02] 0.35+ 0.15 0.09 + 0.02

Varying pH 0.4 ± 0.15 0.074 + 0.02

D 20 Exchange (6 °C) 0.35+ 0.15 0.15 + 0.02

CH4 0.95+ 0.22 0.17 + 0.02 Hexane 0.5 ± 0.15 0.13 + 0.02

Nitrobenzene 0.26± 0.15 0.11 ± 0.02

274.5 K 0.16 + 0.05 0.06 + 0.01

277 K 0.28 + 0.15 0.09 + 0.02

283 K 0.96 + 0.10 0.30 ± 0.02

288 K. 2.6 + 0.3 0.40 ± 0.02

293 K. 6.4 ± 0.4 0.48 + 0.05

298 K 19.7 ± 1.0 1.0 ± 0.1

303 K 53+4 1.8 + 0.3

318 K 132 + 10 8.35 + 1.0 327

- Table 4. Activation Parameters from Arrhenius and Eyring Plots of the Reaction of Hred with Dioxygen. Rate Constant Ea ) AH (kJ) ASt (Jmole 1 K-1)

k2 113 111 147

k3 75 75 8 328

CA

4-

CQ

a. N C.

Ca

co

~L4 329

1- 4 N W,

= r 01:: X

o w V U

oZ o = ._ ; ·e .- ;rrE u

6 :;,.

.:;tl

C)

_ m 330

Figure 2. HPLC traces of single turnover reactions of Hred with nitrobenzene and dioxygen. A control reaction carried out with no NADH is given in trace A. Reactions with one and two equivalents of protein B present are given in traces B and C, respectively. 2-Nitrophenol and 3-nitrophenol would elute where indicated. 331

A.

I.. __ -__ PhNO2

B.

4-nitrophenol "K-4< 2-nitrophenol 3-nitrophenol PhNO2

C.

-_.._. ---4-nitrophenol

PhNO2 332

oCZ

CZo u

t~en a)

N

,._-e

4.a cu x o

-3

X:

._ u 0

o

oO . 0 "

qc;

or,.. 333

\. . ci

~~~pI~ ~ ~~

0 t-~ --- 0 P--

ul)

Tt 4-

00 r_ c1· a)T

. -. Ip,~~~~~~~~·

1 0:I I N -

I1 I !I 0 H In mn o P A °/0 r-.

pTaOT % 334

Figure 4. Plots generated from the catalytic reaction of the complete MMO system with nitrobenzene. The kinetic traces in A were obtained at 404 nm, and the linear portions were used to generate plots B and C. The lag present in A is most likely due inadequate thermal equilibration of the cuvette at 45 °C prior to initiation of the reaction. 335

A.

1

0.8

c) c 0.6

° 0.4

0.2

0

0 50 100 150 200 250 300 Time (sec)

B.

0.005

0.004

M 0.003

0.0023

0.001

0 0 1 2 3 4 5 6 [Substrate] mM

C. y = 92.785 + 486.29x R= 0.98811

I 1

c, 3440

v, < 2580

;: 1720 u ? 860

0 !- I I I I 0 2 4 6 8 1/[Substrate] (mM') 336

Figure 5. Kinetic traces at 404 nm of the reaction of Hred with dioxygen in the absence (trace A) and presence (trace B) of two equivalents of protein B. First order fits are superimposed and yielded rate constants of 0.011 s- l and 0.332 s-l for A and B, respectively. 337

A.

0.024

.. 0.021 rn

0.018

u. 0.015 ©U OCL4 0.01.2

0 60 120 180 240 300 Time (sec)

B.

0.2

0.18

,.6.a *_-4 en 0.16 ©d Cd

._u 0.14

O 0.12

0.1 0 2.4 4.8 7.2 9.6 12 Time (sec) 338

Figure 6. Plot of pseudo-first order rate constant of reactions of Hred with nitrobenzene and dioxygen as a function of the amount of protein B present. The rate constant kobs increases from = 0.011 s- 1 to = 0.36 s- 1. 339

,% 1- U.Z

0.4

'-C' 0.3 CA

-0

0.2

0.1

n 0 0.5 1 1.5 2 2.5 3 3.5 4 Equivalents Protein B 340

Figure 7. (A) Missbauer spectrum in a weak magnetic field (50 mT) of a 155 ms rapid freeze-quench sample. (B) M6ssbauer spectrum for L obtained after subtracting signals for Ho,, Hred, and Q from A. 341

_ · · r j I I

0.0

A. I II I I II 1.0 II Y

C:: I ._C I--, II 0 I I 0 (A -u

0.0

0.5 B.

I

I B I I I I I I I .... i B I - - -4-4 0 4 Velocity (mm/s) 342

Figure 8. Mbssbauer spectrum in a weak magnetic field (50 mT) of a 3 s rapid f:reeze-quench sample. 343

· · r · · I I I I I I I I I I I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 0.0 J1 I0I0poioll 0.2

0.4 I

0.6 _ 0z 0.8 I I 0 I I 1.0 _

1.2 _

1.4

1.6 -

I

=I I I I L I I I I I I -4 -2 0 2 4 VELOCITY (mm/s) 344

Figure 9. (A) Mdssbauer spectrum in a weak magnetic field (50 mT) of a 8 s rapid freeze-quench sample. (B) Mbssbauer spectrum for Q obtained after subtracting signals for Hox, Hred, and L from A. 345

_

0.0 A.

1.0 I II v., I:

- v: 0.0

0.5

I II I I I I -4 0 4 Velocity (mm/s) 346

Figure 10. M/ssbauer spectrum in a weak magnetic field (50 mT) of Hox (A) and

Hred (B) at 4 K. 347

A.

i I I I I I I I I I 0.0 -

0.5

1.0 I I z o 1.5

0 =2.0 I

I I

2.5 I I I II I I

I I I I 3.0

-4 -2 0 2 4 VELOCITY (mm/s) 348

B.

I I I I I I I I I I I

0.0

00010411111

0.5 [P

I lI I I II I I z 1.0 I I-. I 0 I I I I C < 1.5 I I I I I

2.0

I I

I I II , I I I I I I -4 -2 0 2 4 VELOCITY (mm/s) 349

Figure 11. High field M6ssbauer spectra (8 T, 4 K) of Hred (A), L (B), and Q (C). Two species, Hred(l) and Hred(2 ), were found in spectrum A, and a mixture of these gives rise to the spectrum. Simulation of the individual components remains in progress. 350

I I I I I I I I I I I I I I 0.0 0.2 0.4 0.6 0.8

0.0 , 0.2

0z

0 < 0.0 11 0.2 _

= - I I I I I I I I I I -6 -4 -2 0 2 4 6 VELOCITY (mm/s) 351

._

to --

o,-

oCI m

© ' U04 aD- 0

v a 352

Ln

N

0o U

o

an

C;05 (l6suaa Ie;6do XpIsua~j iumid0 353

cr. c

5

C 4-

cnC) C a

'"L ._

_ r

C)

C) tU)

o

C.

-)

C-o *~ 354

o

0

U.) 0 o b.

L- Ioo

0Wm,0 E~

be

LnC,3 X o

CD CDO

too

o 6 n c in r on 0 6 0' 6 0 6 0 aueqlosqv 355

.r a) E

N c)

11 II

tCcu cn

ocZ, co Q

C)

'-a C. .as a3 cn cn

u C co

a) CZ

-C

,.cn C0

*v--

c~ ._0

CD

C1 356

L

C

O CD II Cfo*

'-4

V) V) CIN o

CD

ti In) ' -.

rn

IC In 357

as

,o aC)C)

cnl

,.)0

C)L.) Cl OC. aC)N

C) CD

C) . C)N C.)

ToC.)

C) Cl O4X ._X

C14 358

o

Lo

ef1 0

Ori

m 359

Xa

a 0

0r.

a)

a)c4

cD U u o,

._

0 0 cc a)

a. c-, U r.X 360

0

ro0

O C) rl

o .

LfCU)E

0c, I 0

X1TsuaauJ 361

Figure 17. Resonance Raman spectra of a rapid freeze-quench sample of L from the reaction of Hred with dioxygen (from ref. 33). Excitation was carried out at 647 nm. The inset shows the 860 to 940 cmql region of the spectra obtained from samples made with 1602 and 1802, as well as the difference spectrum. 362

MMO/L intermediate Xo= 647.1 nm

CoDu

ag l l

0 LO CO o C

I . f 1

I I 1000 80)0 600 400 A / -1 LnA/ cm 363

Figure 18. Plots of % M6ssbauer area of the species L (squares), Q (diamonds), and Hox (triangles) over time. Lines represent the fit to equation (4) with k = 28 s1, k2 = 0.46 s-, k3 = 0.026 s-. 364

A.

27

< 20 u

c 14 3

.6.8 :0

0

0 12.8 25.6 38.4 51.2 Time (sec)

B.

" 26

< 19 C.0

c 13

6.4 : 6.4

0 42 8.4 12.6 16.S Time (sec)

C.

' 26

- 19 t, 13 .. n 6.4 :0

0 I I I I I I -1.8 -0.9 0 0.9 1.8 log Time (sec) 365

;

-. aOQ EQ

U

II

cJ- 'T,C;

· c~ua yCUcJ, ·=. aJ °

O ,. a c Q o-

- , O_ o V O,

0) uO U .°0>_

¢ 5 , oO ;Z -

t

._ U

Gt r

._, bD 5 366

o

0

CDIt u 0r)Fv 0c,u e ".

*_

N

r-Co

I co e N Nrq t"co0 i!suaa le'ldo 367

Cr4

"1 od c1 II

Cr

II

C) - .4.)

>-. ct

-,

'-, c.

u0

CQ,) Ce

U~ -rr)

0 4:: U3- ard .%(d.i

C. rJ C. 3-

3- 368

I Ift I

,i Ncrl

H-@- H-U d 6c J , l I .' o e ;0 =~ X

-- ~--- o z en X *1O * I -1 I .,

O 0

w -w I I III .o oNN 0

(I S) lUqlSUOD ao 369

rn a, 0.C

o ca)

a. C)

- ,-o,

> CL)

: C)X

0 _

Cab C

o 370

, i lw -w

I

I i

i

0 i -I

__i: : 0 -w

I i

LO

i tI

[~~Ii I I l~~~~~E~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

o0 D*zt of)0 a 6H C

(ITS) :UIuisuoJ caw}J 371

0 o 0 II

M .11-e

C'4 c-I 0 C/2 o4

CZ

CZ Cl.S c

c.

C) -,

0X

- IC) 1:u 75

xcur S0,-4 ClC14

I- C) ._CD u 1-

1:d .4~J C) .5 C) 0

Cl

- as 0 oo

o C -, . 4

s H

II CI) 372

rll

mo0

oo

0

C

cm of) 0 6° ° ~ 6 r 6 t O Xp!sula leI:ldo 373

Figure 23. An Arrhenius plot (A) and an Eyring plot (B) for the temperature dependence of the rate constants k2 and k3. 374

A. y = 48.152 + -13648x R= 0.99494 I In k2 y = 30.398 + -9060.9x R= 0.98746 - In k3

U

4

2

0

-2

-4 I I I I 0.0031 0.0032 0.0034 0.0035 0.0036 1/T

B. -B.y = 17.657 + -13338x R= 0.99465 y = 0.91047 + -9039.3x R= 0.98752 - k3 -24

-26 I-,

* __ -28

-30 -30

-32

II I I I I I 0.0031 0.0032 0.0034 0.0035 0.0036 1/T 375

U 7 oCa)

uUo ;n

c-I; c5 ,C ::1

O., , 0 a

o C.) ta3 C) En

oz c

.qq .-,as

.

. .. 376

en

es

0o o o o 0 0 0

o c; c;~~o 6 o 6 6 AIsuaa lu:)Tldo 377

n

4--

u

uC

, c "

. 0U4rO

C

a)

I - 1 o 378

0 0 I z I z

n _T 0 I= 0 O owl P- 0 =0 ,IOF 9, \ 1 / _ /0 0 / II: :L 0 Z Z 0 Z O0

I - / - o

0 0 I /z NP7 0

o o 0 0 I Z I z o - W_

1.4 "10 (U O) .4.0 N rd o wr =0 0-0 F -W ::L ed ti 0-4

\o C41

I Z - / 379

Figure 26. Proposed catalytic cycle for the oxidation of hydrocarbons by MMO. See Figure 25 for proposed structures of intermediates L and Q. Substrate first binds to Ho, followed by the reduction of Hox to Hred. Dioxygen binds to Hred and produces intermediate L. Rearrangement, which may include proton transfer, yields intermediate Q. Substrate then reacts with Q to produce alcohol,

1H20, and Hox. 380

R-H-- ROH + H20 FeI',O Fe"'

H -1

H20 2 R-H R-H Fe"Io" Fe"'

shunt H pathway

0.31 s q1 NADH j + 2H R-H NAD'.. T+H' w,-. J. Fe2 III(2 -2) I eIIo FeII w"L' 2 25 Sl R-H H

02 381

Biography

Katherine Liu was born in Dayton, Ohio, on September 28, 1967. She spent her childhood mainly in suburban Detroit, but moved with her family to South Bend, Indiana for a brief period. Kathy attended Cornell University in Ithaca, New York, where she obtained her B. A., majoring in chemistry. At Cornell, Kathy was introduced to chemical research in the laboratory of Dr. 1H-ctor D. Abrufia. After graduating in 1989, she enrolled in the graduate program in chemistry at the Massachusetts Institute of Technology in Cambridge, Massachusetts. Her thesis work at MIT was carried out in the laboratory of Dr. Stephen J. Lippard, and focused on the mechanistic aspects of the enzymatic system methane monooxygenase. Kathy completed her Ph.D. in February, 1995.