Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.

The dinitrogen-binding site in the Mo-based nitrogenase is FeMo-cofactor, a metallo-sulfur cluster of composition MoFe7S9·R-homocitrate. The NifV- mutant nitrogenase from Klebsiella pneumoniae contains an FeMo-cofactor in which homocitrate has been replaced by citrate (i.e., MoFe7S9·citrate). Both the wild type and mutant cofactors (in the S = 3/2 spin state) can be extracted into N-methylformamide. The extracted cofactors bind one molecule of PhS- at the tetrahedral Fe, and the rate of this reaction depends on what else is coordinated to the cluster. No differences were observed between the reactivities of wild-type and NifV- cofactors with PhS- when they were complexed with CN-, N3 -, or H+. However, when imidazole is bound, the kinetics of the reactions of PhS- with the two cofactors are very different. Here we propose that R-homocitrate (but not citrate) can hydrogen bond to the imidazole ligand on Mo, and that this perturbs the electron distribution within the cluster core, and hence its reactivity with PhS-. Using the X-ray crystallographic data for the MoFe-protein of nitrogenase and molecular mechanics calculations, we have investigated the implications of these findings on the action of the enzyme. Our model shows that R-homocitrate is uniquely capable of facilitating the binding of dinitrogen by allowing the substrate access to Mo after dissociation of the Mo−carboxylate bond while simultaneously influencing the electron-richness of the cofactor by hydrogen bonding of the pendant −CH2CH2CO2 arm to the imidazole group of Hisα442. The whole process is mediated by hydrogen bonding of amino acid side chains to the carboxylate groups of R-homocitrate.

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