| Abstract
| - Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to the hydroxylasecomponent (MMOH) of soluble methane monooxygenase (sMMO) primes its non-heme diiron centers forreaction with dioxygen to generate high-valent iron intermediates that convert methane to methanol. Thisintermolecular electron-transfer step is facilitated by a reductase (MMOR), which contains [2Fe-2S] andflavin adenine dinucleotide (FAD) prosthetic groups. To investigate interprotein electron transfer, chemicallyreduced MMOR was mixed rapidly with oxidized MMOH in a stopped-flow apparatus, and optical changesassociated with reductase oxidation were recorded. The reaction proceeds via four discrete kinetic phasescorresponding to the transfer of four electrons into the two dinuclear iron sites of MMOH. Pre-equilibratingthe hydroxylase with sMMO auxiliary proteins MMOB or MMOD severely diminishes electron-transferthroughput from MMOR, primarily by shifting the bulk of electron transfer to the slowest pathway. Thebiphasic reactions for electron transfer to MMOH from several MMOR ferredoxin analogues are also inhibitedby MMOB and MMOD. These results, in conjunction with the previous finding that MMOB enhances electron-transfer rates from MMOR to MMOH when preformed MMOR−MMOH−MMOB complexes are allowed toreact with NADH [Gassner, G. T.; Lippard, S. J. Biochemistry1999, 38, 12768−12785], suggest thatisomerization of the initial ternary complex is required for maximal electron-transfer rates. To account forthe slow electron transfer observed for the ternary precomplex in this work, a model is proposed in whichconformational changes imparted to the hydroxylase by MMOR are retained throughout the catalytic cycle.Several electron-transfer schemes are discussed with emphasis on those that invoke multiple interconvertingMMOH populations.
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