KINETIC AND SPECTROSCOPIC STUDIES OF COBALT- AND MANGANESE- SUBSTITUTED EXTRADIOL-CLEAVING HOMOPROTOCATECHUATE 2,3- DIOXYGENASES A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Andrew J. Fielding IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Professor Lawrence Que, Jr. February 2013 © Andrew Jay Fielding 2013 Acknowledgements I would like to thank all those who have helped me in my efforts here at graduate school at the University of Minnesota including: My advisors Professors Larry Que and John Lipscomb for their instruction and advice. I would also like to thank my comrades in the Que and Lipscomb labs, specifically Drs. Erik Farquhar, Van Vu, and Mike Mbughuni, and fellow graduate students Rahul Banerjee, Brent Rivard, and Anna Komor for their friendship and the time they spent helping me. I would also like to thank all the many postdoctoral research associates from the Que and Lipscomb labs who have taken time to talk to me about my chemistry including: Ass’t Prof. Joseph Emerson, Dr. Elena Kovaleva, Ass’t Prof. Tom Makris, Dr. Yuming Zhou, Ass’t Prof. Aidan McDonald, Ass’t Prof. Matthew Cranswick, Ass’t Prof. Kathy Van Heuvelen, Dr. Williamson Oloo and Dr. Lisa Engstrom. I am also grateful for the generous support of the University of Minnesota Department of Chemistry, the University of Minnesota Chemical Biology Initiative and the National Institutes of Health. I would also like to thank my parents Kurt and Patti Fielding who encouraged my interests in Science while growing up, where dinner table discussions regularly lead to referencing our copy of the Encyclopedia Britannica . I would also like to thank my many terrific chemistry and biology teachers throughout junior high, high school and college for making science fun and interesting as well as training me to think like a scientist. Finally I would like to give special thanks to my wife Meikjn and our kids for their support and giving my life and work meaning. i Abstract Homoprotocatechuate (HPCA) 2,3-dioxygenase (HPCD) is an Fe(II)-dependent extradiol-cleaving dioxygenase, which oxidatively cleaves the aromatic C(2)-C(3) bond of its catecholic substrate. Here we compare the reactivity of Fe-HPCD with its Mn(II)- and Co(II)-substituted analogues. While Mn-HPCD exhibits steady-state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD exhibits significantly higher O2 KM and kcat values. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. These kinetic differences and the spectroscopic properties of Co-HPCD have proven to be useful in further exploring the unique O 2 activation mechanism associated with the extradiol dioxygenase family. Employing the electron-poor substrate analogue 4-nitrocatechol (4NC), which is expected to slow down the rate of catechol oxidation, we were able to trap and characterize the initial O2-adduct in the single-turnover reaction of 4-nitrocatechol by Co-HPCD. This intermediate exhibits an S = 1/2 EPR signal typical of low-spin Co(III)−superoxide complexes. Both the formation and decay of the low-spin Co(III)−superoxide intermediate are slow compared to the analogous steps for turnover of 4NC by native high-spin Fe(II)-HPCD, which is likely to remain high-spin upon O 2 binding. Possible effects of the observed spin-state transition upon the rate of O 2 binding and catechol oxidation are discussed. Two transient intermediates were detected in the reaction of the [M-HPCD(4XC)] enzyme-substrate complexes (M = Mn or Co, and 4XC = 4-halocatechols, where X = F, ii 4XlC Cl, and Br) with O 2. The first intermediate (Co Int1) exhibited an S = 1/2 EPR signal associated with an organic radical species. Based on the UV-Vis and EPR data, Co 4XC Int1 was assigned to a unique low-spin [Co(III)(4XSQ •)(hydro)peroxo] species where the semiquinone radical is localized onto C4 of the ring. M4XC Int2 was observed to have a high-spin metal(II) center by EPR and exhibit intense chromophores similar to the independently synthesized halogenated quinones (4XQ). Based on the UV-Vis and EPR data, M 4XC Int2 is assigned to a [M(II)(4XQ)(hydro)peroxo] species. The M4XC Int2 species were further characterized by resonance Raman spectroscopy. Resonance enhanced vibrations between 1350–1450 cm -1 suggest that M4XC Int2 is a metal-semiquinone species, conflicting with the initial assignment of these intermediates as a quinone species. Based on the EPR and resonance Raman data, M4XC Int2 might be • •− assigned to a [M(II)(SQ )O 2 ] diradical species. iii Table of Contents Acknowledgments i Abstract ii Table of Contents iv List of Tables ix List of Figures xi List of Schemes xxxiii Chapter 1 O2 Activation and Substrate Oxidation Mechanisms of C-C Bond Cleaving Oxygenases and Oxidases 1 1.1 Introduction 1 1.2 Reductive O 2 activation and substrate oxidation 9 1.3 Metal cofactor and active sites 9 1.4 Common and divergent mechanistic themes 13 1.4.1 Organic substrate binding and activation 13 1.4.2 Reductive O 2 activation and substrate oxidation 14 1.4.3 Reductive O 2 activation by a metal cofactor 17 1.4.4 Mechanism of substrate oxidation 17 1.4.5 Reactive pairs 19 1.4.6 Formation of an alkylperoxo or (hydro)peroxo intermediate 19 1.4.7 Rearrangement of the alkylperoxo or (hydro)peroxo intermediate 22 1.4.8 Product formation and release 26 1.5 Mechanistic details of representative C-C cleaving dioxygenases and oxidases 26 1.5.1 Extradiol-cleaving catechol dioxygenases 27 1.5.2 Metal substitution experiments 28 1.5.3 Proposed mechanisms of reductive O 2 activation and substrate oxidation 31 1.5.4 Trapped O 2 intermediates 32 1.5.5 Intermediates trapped with second sphere mutants 33 1.5.6 DFT calculations 36 1.5.7 Mechanism of alkylperoxide rearrangement 39 1.6 Aim and scope of thesis 43 Chapter 2 A Hyperactive Cobalt-Substituted Extradiol-Cleaving 44 Catechol Dioxygenase 2.1 Introduction 45 2.2 Materials and methods 49 2.2.1 Reagents and general procedures 49 2.2.2 Preparation of M(II)-HPCD 49 2.2.3 Molar absorptivity of Co-HPCD 49 iv 2.2.4 ICP-AES metal analysis 49 2.2.5 Steady-state kinetics 49 2.2.6 Activation and inhibition studies 50 2.2.7 Transient kinetic experiments 51 2.2.8 X-ray crystallography 51 2.2.9 UV-Vis absorption spectroscopy 52 2.2.10 EPR sample preparation and spectroscopic methods 53 2.3 Results 53 2.3.1 Metal-substituted enzymes 53 2.3.2 Activation and inhibition studies 56 2.3.3 Steady-state kinetics 58 2.3.4 X-ray crystal structure comparisons 60 2.3.5 Spectroscopic characterization 63 2.3.5.A Electronic absorption spectra of high-spin Co(II) enzyme and enzyme-substrate complexes 63 2.3.5.B EPR spectra of high-spin Co(II) enzyme and enzyme-substrate complexes 65 2.3.5.C Electronic absorption spectra of enzyme complexes with wt- and H200N-Co-HPCD 69 2.3.5.D X-ray absorption spectroscopy studies of Co-HPCD complexes 73 2.3.5.E Electron paramagnetic resonance spectra of 3HPA enzyme- inhibitor complexes 75 2.3.6 Transient kinetic experiments 76 2.4 Discussion 78 2.4.1 Mechanism proposed for extradiol dioxygenases 78 2.4.2 Basis for the high turnover rate of Co-HPCD 80 2.4.3 Effect of Co(II) substitution on reaction cycle rate constants 82 2.4.4 Effects of Co(II) substitution on HPCD catalysis 85 2.5 Supporting information: X-ray crystallographic studies 86 Chapter 3 Characterization of a an O 2 Adduct of an Active Cobalt-Substituted Extradiol-Cleaving Dioxygenase 96 3.1 Introduction 96 3.2 Experimental methods 96 3.2.1 Reagents 96 3.2.2 Preparation of metal-substituted wt-HPCD and H200N-HPCD 97 3.2.3 EPR sample preparation and spectroscopic methods 97 3.2.4 Determination of the dissociation constant for 4NC 98 3.2.5 Kinetic experiments 98 3.2.6 Global analysis 99 3.2.7 pH-Activity profiles 100 3.2.8 EPR freeze-quench kinetic experiments 100 3.2.9 Spin quantification 101 v 3.3 Experimental results 101 3.3.1 Turnover of 4NC 101 3.3.2 O2 concentration dependence 103 3.3.3 EPR freeze-quench experiments 105 3.4 [H200N-Co-HPCD(4NC)superoxide] 110 3.4.1 Reversible O 2 binding 110 17 3.4.2 O2 EPR experiment 114 3.5 Low-spin to high-spin transition 118 3.6 Role of H200 119 3.6.1 [wt-Co-HPCD(4NC)] and [H200N-Co-HPCD(4NC)] pH titrations 120 3.6.2 Effect of H200N mutation 124 3.7 Summary 125 3.8 Inactivity of [Mn-HPCD(4NC)], [H200N-Mn-HPCD(4NC)], and [H200N- Mn-HPCD(HPCA)] complexes toward O 2 binding. 126 3.9 Nitric oxide binding to Co-HPCD and Mn-HPCD enzyme-substrate complexes. 128 3.10 Possible explanation for unexpected differences in reactivity of Mn and Co-HPCD. 130 3.11 Inactivity of Co-H200Q 131 Chapter 4 Kinetic and Spectroscopic Experiments Characterizing Intermediate Species Observed in the Reaction of [Co-HPCD(4XC)] with O 2 (4XC = 4-Halogenated Catechols, X = F, Cl or Br). 133 4.1 Introduction 133 4.2 Results 139 4.2.1 Steady-state kinetics 139 4.2.1.A Inactivation of Fe-HPCD during turnover of 4ClC 139 4.2.1.B Steady-state kinetic parameters 143 4.2.1.C Linear free-energy steady-state studies 144 4.2.1.D Temperature dependence of steady-state parameters 145 4.2.1.E pH activity profiles 145 4.2.2 Transient kinetic experiments 148 4.2.2.A Stopped-flow experiments 148 4.2.2.B O 2 concentration dependence 153 4.2.2.C Solvent kinetic isotope effect 153 4.2.2.D Temperature dependence of rates 154 4.2.3 Spectroscopic characterization 156 4.2.3.A X-band EPR spectra of wt- and H200N-Co-HPCD enzyme- substrate complexes 156 4.2.3.B Rapid freeze-quench experiments: [Co-HPCD(4XC)] rapidly