Bioinorganic Hydrocarbon Oxidation : Mechanistic and Kinetic Studies

Chapter 1. Principles of Small Molecule Activation by Metalloproteins as Exemplified by the Soluble Methane Monooxygenase Many metalloenzymes activate small molecules in a manner that is unique to natural systems. In this perspective we discuss the soluble protein system from Methylococcus capsulatus (Bath), which uses a mixed-function hydroxylase to convert methane selectively to methanol. Through a series of biophysical studies, theoretical calculations, synthetic model studies, and mechanistic biochemical experiments, the respective roles of the carboxylatebridged non-heme diiron center and the protein environment in controlling the enzyme mechanism have been delineated. These results are used to identify themes common among metalloenzymes that activate small molecules and to identify future directions for the study of this protein system. Chapter 2. Small Molecule Binding to the Mixed-Valent Diiron Center of Methane Monooxygenase Hydroxylase from Methylococcus capsulatus (Bath) as Revealed by ENDOR Spectroscopy The binding of several exogenous molecules at the active site of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) is described. The interaction of methanol, the product of methane hydroxylation, with the mixed-valent FeIIFe III form of the enzyme induces a change in the EPR spectrum of the unmodified protein from g = 1.940, 1.865, and 1.740 to one with gi = 1.963 and g2 = 1.862. This spectral change arises from coordination of methanol to the diiron center, as demonstrated by ENDOR spectroscopic studies of mixed-valent MMOH prepared with CD 30D added to a DMSO-treated sample, which displayed a 2H interaction with an isotropic hyperfine splitting of ~0.5 MHz. A detailed analysis indicates that methanol binds to the ferrous iron without displacing a terminally bound water molecule identified previously. Acetate labeled with 13C at the carboxylate carbon atom also gives rise to ENDOR signals, whereas no such signals are seen when the label is at the methyl carbon atom or when the methyl group is deuterated. These results suggest that acetate is oriented in the active site with its carboxylate group pointing towards the iron center, confirming a previous interpretation of X-ray crystallographic experiments. Four coordination positions at the MMOH diiron core not usually occupied by endogenous protein ligands are identified which can simultaneously accommodate DMSO, methanol, and water. The implications of these results for the catalytic mechanism of the enzyme are discussed. Chapter 3. An EPR Study of the Dinuclear Iron Site in the Soluble Methane Monooxygenase Reduced by One Electron at 77 K: the Effect of Component Interactions and the Binding of Small Molecules to the Dinuclear Ferric Center EPR spectra of cyrogenically reduced diferric sites from the soluble methane monooxygenase hydroxylase (MMOH) were examined as probes of active site structure. In the absence of exogenous ligands and coupling protein (MMOB), two populations of active sites were observed, one species (H1) having g values of 1.94, 1.86, and 1.79 and the other more anisotropic signal (H2) having g values of 1.82, 1.77, and 1.68. These forms can be distinguished by the temperatureand power-dependence of their EPR signal. H1 and H2 are postulated to be related by proton movements, and they interact differently with small exogenous molecules. Some interactions, such as that of DMSO with the oxidized MMOH, are mediated by MMOB. Additional species afforded by small molecule and protein binding are interpreted according to this model, with additional modifications afforded by carboxylate shifts of a flexible glutamate ligand. Chapter 4. An Investigation of the Reaction of Diferrous Methane Monooxygenase Hydroxylase with Dioxygen and Substrates by Rapid FreezeQuench and Stopped-Flow Spectroscopy Single turnover reactions of MMOH are studied in the presence and absence of substrate and by continuous and discontinuous techniques. The electronic absorption spectrum (Xmax = 725 nm, F = 1500 M-1 cm-1) of the Hperoxo intermediate is presented for the first time and activation parameters for its formation are reported. The rate constant for formation of Hperoxo is not dioxygen concentration dependent. Activation parameters for the decay of Hperoxo and the formation of Q are in good agreement with each other. Photoreactivity of intermediate Q is described, and the mechanism of its decay in the absence of substrate studied. Irradiation of a frozen solution of Q by y rays produces a new paramagnetic species, Qx, which is spectroscopically similar to intermediate X in ribonucleotide reductase R2 cofactor assembly. The M6ssbauer characterization of Qx is reported. A new oxo-bridged diferric species is detected by rapid freeze-quench (RFQ) M6ssbauer. Finally, the reactions of substrates with intermediate species are studied by using RFQ M6ssbauer and single and double mixing stopped flow spectroscopy. The data suggest that Hperoxo or a related species other than Q is competent to oxidize olefins. The reactions of substrates with Q are studied. The data support a more complicated mechanism than a simple bimolecular collision, with the reaction order in substrate decreasing with increasing substrate size.