Exploring the reactivity of bacterial multicomponent monooxygenases

Abstract

Chapter 1. Introduction: The Reactivity of Bacterial Multicomponent Monooxygenases Bacterial multicomponent monooxygenases constitute a remarkable family of enzymes that oxidize small, inert hydrocarbon substrates using molecular oxygen. Three or more protein components are required for the timely reactions of electrons, protons, 02, and hydrocarbon at an active site carboxylate-bridged diiron center. This overview describes structural and biochemical studies of the BMM protein components, presents the proposed mechanisms of 02 activation by BMMs and related carboxylate-bridged diiron proteins, and discuses substrate reactivity of the oxygenated diiron units responsible for BMM catalysis. Chapter 2. Revisiting the Mechanism of Dioxygen Activation in Soluble Methane Monooxygenase from M. capsulatus (Bath): Evidence for a Multi-Step, Proton- Dependent Reaction Pathway Stopped-flow kinetic investigations of soluble methane monooxygenase (sMMO) from M. capsulatus (Bath) have clarified discrepancies that exist in the literature regarding several aspects of catalysis by this enzyme. The development of thorough kinetic analytical techniques has led to the discovery of two novel oxygenated iron species that accumulate in addition to the well-established intermediates Hroxo and Q. The first intermediate, P*, is a precursor to Hperoxo and was identified when the reaction of reduced MMOH and MMOB with 02 was carried out in the presence of a540 mM methane to suppress the dominating absorbance signal due to Q. The optical properties of P* are similar to those of H...O, with e420 = 3500 M cm- and F72O = 1250 M cm-. These values are suggestive of a peroxo-to-iron(III) charge-transfer transition and resemble those of peroxodiiron(III) intermediates characterized in other carboxylate-bridged diiron proteins and synthetic model complexes. The second identified intermediate, Q*, forms on the pathway of Q decay when reactions are performed in the absence of hydrocarbon substrate. Q* does not react with methane, forms independently of buffer composition, and displays a unique shoulder at 455 nm in its optical spectrum. Studies conducted at different pH values reveal that rate constants corresponding to P* decay/Hpeo formation and H,,,, decay/Q formation are both significantly retarded at high pH and indicate that both events require proton transfer. The processes exhibit normal kinetic solvent isotope effects (KSIEs) of 2.0 and 1.8, respectively, when the reactions are performed in D20. Mechanisms are proposed to account for the observations of these novel intermediates and the proton dependencies of P* to Hroxo and Hproxo to Q conversion. Chapter 3. Oxidation Reactions Performed by Soluble Methane Monooxygenase Hydroxylase Intermediates Hroxo and Q Proceed by Distinct Mechanisms Soluble methane monooxygenase is a bacterial enzyme that converts methane to methanol at a carboxylate-bridged diiron center with exquisite control. Because the oxidizing power required for this transformation is demanding, it is not surprising that the enzyme is also capable of hydroxylating and epoxidizing a broad range of hydrocarbon substrates in addition to methane. In this work we take advantage of this promiscuity of the enzyme to gain insight into the mechanisms of action of Hperoxo and Q, two oxidants that are generated sequentially during the reaction of reduced protein with 02. Using double-mixing stopped flow spectroscopy, we investigate the reactions of the two intermediate species with a panel of substrates of varying C-H bond strength. Three classes of substrates are identified according to the rate-determining step in the reaction. We show for the first time that an inverse trend exists between the rate constant of reaction with HPro,, and the C-H bond strength of the hydrocarbon examined for those substrates in which C-H bond activation is rate-limiting. Deuterium kinetic isotope effects reveal that reactions performed by Q, but not Hroxo, involve extensive quantum mechanical tunneling. This difference sheds light on the observation that Hrox is not a potent enough oxidant to hydroxylate methane, whereas Q can perform this reaction in a facile manner. In addition, the reaction of Hperoxo with acetonitrile appears to proceed by a distinct mechanism in which a cyanomethide anionic intermediate is generated, bolstering the argument that Hroxo is an electrophilic oxidant and operates via twoelectron transfer chemistry. Chapter 4. Dioxygen Activation and the Multiple Roles of Component Proteins in Phenol Hydroxylase from Pseudomonas sp. OX1 02 activation was also investigated in PH, a BMM that oxidizes phenol to catechol. Rapid freeze-quench M6ssbauer and stopped-flow optical spectroscopy were employed to study the reaction of the reduced, diiron(II) form of Pseudomonas sp. OXi PH hydroxylase (PHH) with 02 in the presence of the regulatory protein PHM. A single longlived diiron(III) intermediate with 6 = 0.59 mm/s and A EQ = 0.63 mm/s and no discernable optical bands accumulates along the reaction pathway. The spectroscopic parameters of this intermediate are similar to those reported recently for a diiron(III) transient generated in toluene/o-xylene monooxygenase hydroxylase but are quite different from those of peroxodiiron(III) species formed in other diiron enzymes despite the fact that the active sites of these proteins have identical first-shell coordination environments. In contrast to reactions of MMOH, there is no evidence for accumulation of a high-valent diiron(IV) intermediate in PHH. Under steady state conditions in the absence of hydrocarbon substrate, electrons are consumed and PHH generates H20 2 catalytically, suggesting that the observed diiron(III) intermediate is a peroxodiiron(III) species. Steady state biochemical studies were conducted to ascertain the functions of the PH reductase and regulatory protein. Single turnover experiments revealed that, unlike sMMO, only the complete system containing all three protein components is capable of oxidizing phenol. The yield of catechol produced under ideal conditions maximized at -50% per diiron active site in single turnover experiments, suggesting that the enzyme operates by a half-sites reactivity mechanism. Results from single turnover studies in which the oxidized form of the reductase, PHP, was added to a mixture of reduced hydroxylase and regulatory protein revealed that PHP exerts an additional regulatory effect on PHH, most likely by an allosteric mechanism. The rate of H20 2 formation by PHH in the absence of a hydrocarbon substrate was retarded when PHM was omitted from the reaction mixture, indicating that the regulatory protein controls the kinetics of 02 activation and/or blocks unproductive quenching of the oxygenated intermediate by untimely electron transfer. Chapter 5. Characterization of Iron Dinitrosyl Species Formed in the Reaction of Nitric Oxide with a Biological Rieske Center Reactions of nitric oxide with cysteine-ligated iron-sulfur cluster proteins typically result in disassembly of the iron-sulfur core and formation of dinitrosyl iron complexes (DNICs). Here we report the first evidence that these species can also form at Riesketype [2Fe-2S] clusters. Upon treatment of a Rieske protein, component C of toluene/oxylene monooxygenase (ToMOC) from Pseudomonas sp. OXI, with NO (g) or the NOgenerators S-nitroso-N-acetyl-DL-pencillamine (SNAP) and diethylamine NONOate (DEANO), the absorbance features of the [2Fe-2S] cluster bleach and a new band slowly appears at 367 nm. Characterization of the reaction products by EPR, M6ssbauer, and NRVS spectroscopy reveals that the primary product observed in the reaction is the dinuclear iron dinitrosyl Roussin's red ester (RRE), [Fe2(g-SCys) 2(NO)4], and that mononuclear DNICs only account for a minor fraction of the nitrosylated iron. The RRE reaction product can be reduced by sodium dithionite to produce the one-electron reduced Roussin's red ester (rRRE) having absorption bands at 640 and 960 nm. These results show that NO reacts readily with protein-based Rieske centers and suggest that dinuclear RRE species, not mononuclear DNICs, may be the primary iron dinitrosyl species responsible for the pathological and physiological effects of nitric oxide in the presence of iron-sulfur clusters. Appendix A. Preliminary Characterization of ''Fe-enriched MMOH. and MMOH by Nuclear Vibrational Resonance Spectroscopy Synchrotron-based 57Fe Nuclear Resonance Vibrational Spectroscopy (NRVS) is a powerful technique that allows for identification of the full set of vibrational modes of a given "Fe center. In this work we present preliminary NRVS studies of 57Fe-enriched oxidized soluble methane monooxygenase hydroxylase in complex with 2 equiv of its regulatory protein (MMOH.x:2B) and intermediate Q, the species responsible for methane oxidation in this enzyme. Although maximal protein concentrations were employed, very few vibrational peaks were resolved. Our data suggest that the sMMO protein system is not amenable to this method using the technologies that are currently available.