Mechanistic studies of electron transfer, complex formation, C-H bond activation, and product binding in soluble methane monooxygenase

Abstract

Chapter 1. Soluble Methane Monooxygenase: Activation of Dioxygen and Methane The mechanisms by which soluble methane monooxygenase uses dioxygen to convert methane selectively to methanol have come into sharp focus. Diverse techniques have clarified subtle details about each step in the reaction, from binding and activating dioxygen, to hydroxylation of alkanes and other substrates, to the electron transfer events required to complete the catalytic cycle. Chapter 2. Electron Transfer Reactions of the Reductase Component of Soluble Methane Monooxygenase from Methylococcus capsulatus (Bath) Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear non-heme iron active site, a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH, and a coupling protein (MMOB). MMOR uses a non-covalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes ...

(cont.) The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- s-1 for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system. Chapter 3. Structural Features of the MMOH/MMOR Complex as Revealed by Mass Spectrometric Analysis of Covalently Cross-linked Proteins. Soluble methane monooxygenase requires complexes between its three component proteins for efficient catalytic turnover. The hydroxylase (MMOH) must bind both to the reductase (MMOR) for electron transfer and to the regulatory protein (MMOB) to allow reaction with substrates. Although structures of MMOH, MMOB, and one domain of MMOR have been determined, little is known about structures of the complexes. Proteins cross-linked by a carbodiimide reagent were analyzed by specific proteolysis and capillary HPLC-mass spectrometry. Tandem mass spectra conclusively identified two amine-to-carboxylate cross-linked sites involving the alpha subunit of MMOH and the [2Fe-2S] domain of MMOR (MMOR-Fd). The amino terminus of the MMOH alpha subunit cross-links to the side chains of MMOR-Fd residues Glu56 and Glu91. These Glu residues are close to one another on the surface of MMOR-Fd and far from the [2Fe-2S] cluster ...