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Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to the hydroxylase component (MMOH) of soluble methane monooxygenase (5MMO) primes its non-heme diiron centers for reaction with dioxygen to generate high-valent iron intermediates that convert methane to methanol. This intermolecular electron-transfer step is facilitated by a reductase (MMOR), which contains [2Fe-2S] and flavin adenine dinucleotide (FAD) prosthetic groups. To investigate interprotein electron transfer, chemically reduced MMOR was mixed rapidly with oxidized MMOH in a stopped-flow apparatus, and optical changes associated with reductase oxidation were recorded. The reaction proceeds via four discrete kinetic phases corresponding to the transfer of four electrons into the two dinuclear iron sites of MMOH. Pre-equilibrating the hydroxylase with sMMO auxiliary proteins MMOB or MMOD severely diminishes electron-transfer throughput from MMOR, primarily by shifting the bulk of electron transfer to the slowest pathway. The biphasic reactions for electron transfer to MMOH from several MMOR ferredoxin analogues are also inhibited by 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 to react with NADH [Gassner, G. T.; Lippard, S. J. Biochemisti'y 1999, 38, 12768-12785], suggest that isomerization of the initial ternary complex is required for maximal electron-transfer rates. To account for the slow electron transfer observed for the ternary precomplex in this work, a model is proposed in which conformational 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 interconverting MMOH populations. [ABSTRACT FROM AUTHOR]

The de novo protein DR is a minimal model for diiron and dimanganese metalloproteins, such as soluble methane monooxygenase. DF1 is a homodimeric four-helix bundle whose dinuclear center is formed by two bridging Glu side chains, two chelating Glu side chains, and two monodentate His ligands. Here, we report the di-Mn(ll) and di-Co(ll) derivatives of variants of this protein. Together with previously solved structures, 23 crystallographically independent four-helix bundle structures of DF1 variants have been determined, which differ in the bound metal ions and size of the active site cavity. For the di-Mn(ll) derivatives, as the size of the cavity increases, the number and polarity of exogenous ligands increases. This collection of structures was analyzed to determine the relationship between protein conformation and the geometry of the active site. The primary mode of backbone movement involves a coordinated tilting and sliding of the first helix in the helix-loop-helix motif. Sliding depends on crystal-packing forces, the steric bulk of a critical residue that determines the dimensions of the active site access cavity, and the intermetal distance. Additionally, a torsional motion of the bridging carboxylates modulates the intermetal distance. This analysis provides a critical evaluation of how conformation, flexibility, and active site accessibility affect the geometry and ligand-binding properties of a metal center. The geometric parameters defining the DF structures were compared to natural diiron proteins; DF proteins have a restricted active site cavity, which may have implications for substrate recognition and chemical stability. [ABSTRACT FROM AUTHOR]