Structural features of covalently cross-linked hydroxylase and reductase proteins of soluble methane monooxygenase as revealed by mass spectrometric analysis.

Soluble methane monooxygenase requires complexes between its three component proteins for efficient catalysis. The hydroxylase (MMOH) must bind both to the reductase (MMOR) and to the regulatory protein (MMOB) to facilitate oxidation of methane to methanol. Although structures of MMOH, MMOB, and one domain of MMOR have been determined, less geometric information is available for the complexes. To address this deficiency, MMOH and MMOR were cross-linked by a carbodiimide reagent and analyzed by specific proteolysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and capillary high performance liquid chromatography 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). In particular, the N terminus of the MMOH alpha subunit forms cross-links to the side chains of MMOR-Fd residues Glu-56 and Glu-91. These Glu residues are close to one another on the surface of MMOR-Fd and >25 A from the [2Fe-2S] cluster. Because the N terminus of the alpha subunit of MMOH was not located in the crystal structure of MMOH, a detailed structural model of the complex based on the cross-link was precluded; however, a previously proposed binding site for MMOR on MMOH could be ruled out. Based on the cross-linking results, a MMOR E56Q/E91Q double mutant was generated. The mutant retains >80% of MMOR NADH oxidase activity but reduces sMMO activity to approximately 65% of the level supported by the wild type reductase. Cross-linking to MMOH was diminished but not abolished in the double mutant, indicating that other residues of MMOR also form cross-links to MMOH.

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.

Evaluation of norcarane as a probe for radicals in cytochome p450- and soluble methane monooxygenase-catalyzed hydroxylation reactions.

Norcarane was employed as a mechanistic probe in oxidations catalyzed by hepatic cytochome P450 enzymes and by the soluble methane monooxygenase (sMMO) enzyme from Methylococcuscapsulatus (Bath). In all cases, the major oxidation products (>75%) were endo- and exo-2-norcaranol. Small amounts of 3-norcaranols, 2-norcaranone, and 3-norcaranone also formed. In addition, the rearrangement products (2-cyclohexenyl)methanol and 3-cycloheptenol were detected in the reactions, the former possibly arising from a radical intermediate and the latter ascribed to a cationic intermediate. The formation of the cation-derived rearrangement product is consistent with one or more reaction pathways and is in accord with the results of previous probe studies with the same enzymes. The appearance of the putative radical-derived rearrangement product is in conflict with other mechanistic probe results with the same enzymes. The unique implication of a discrete radical intermediate in hydroxylations of norcarane may be the consequence of a minor reaction pathway for the enzymes that is not manifest in reactions with other probes. Alternatively, it might reflect a previously unappreciated reactivity of norcaranyl cationic intermediates, which can convert to (2-cyclohexenyl)methanol. We conclude that generalizations regarding the intermediacy of radicals in P450 and sMMO enzyme-catalyzed hydroxylations based on the norcarane results should be considered hypothetical until the origin of the unanticipated results can be determined.

Product binding to the diiron(III) and mixed-valence diiron centers of methane monooxygenase hydroxylase studied by (1,2)H and (19)F ENDOR spectroscopy.

The binding of ethanol and 1,1,1-trifluoroethanol (TFE) to both the H(mv) and H(ox) forms of soluble methane monooxygenase (sMMO) in solution has been studied by Q-band (35 GHz) CW and pulsed ENDOR spectroscopy of (1)H, (2)H and (19)F nuclei of exogenous ligands. As part of this investigation we introduce (19)F, in this case from bound TFE, as a new probe for the binding of small molecules to a metalloenzyme active site. The H(mv) form was prepared in solution by chemical reduction of H(ox). For study of H(ox) itself, frozen solutions were subjected to gamma-irradiation in the frozen solution state at 77 K, which affords an EPR-visible mixed-valent diiron center, denoted (H(ox))(mv), held in the geometry of the diiron(III) state. The (19)F and (2)H ENDOR spectra of bound TFE together with (1,2)H ENDOR spectra of bound ethanol indicate that the alcohols bind close to the Fe(II) ion of the mixed-valence cluster in H(mv) and in a bridging or semi-bridging fashion to H(ox). DMSO does not affect the binding of either of the ethanols or of methanol to H(ox), nor of ethanol or methanol to H(mv). It does, however, displace TFE from the diiron site in H(mv). These results provide the first evidence that crystal structures of sMMO hydroxylase into which product alcohols were introduced by diffusion represent the structures in solution.

Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins A list of abbreviations can be found in Section 7.

Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O(2). The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.

Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins.

Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O2 . The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.