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

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