Expression and association of group IV nitrogenase NifD and NifH homologs in the non-nitrogen-fixing archaeon Methanocaldococcus jannaschii.

Using genomic analysis, researchers previously identified genes coding for proteins homologous to the structural proteins of nitrogenase (J. Raymond, J. L. Siefert, C. R. Staples, and R. E. Blankenship, Mol. Biol. Evol. 21:541-554, 2004). The expression and association of NifD and NifH nitrogenase homologs (named NflD and NflH for "Nif-like" D and H, respectively) have been detected in a non-nitrogen-fixing hyperthermophilic methanogen, Methanocaldococcus jannaschii. These homologs are expressed constitutively and do not appear to be directly involved with nitrogen metabolism or detoxification of compounds such as cyanide or azide. The NflH and NflD proteins were found to interact with each other, as determined by bacterial two-hybrid studies. Upon immunoisolation, NflD and NflH copurified, along with three other proteins whose functions are as yet uncharacterized. The apparent presence of genes coding for NflH and NflD in all known methanogens, their constitutive expression, and their high sequence similarity to the NifH and NifD proteins or the BchL and BchN/BchB proteins suggest that NflH and NflD participate in an indispensable and fundamental function(s) in methanogens.

The natural history of nitrogen fixation.

In recent years, our understanding of biological nitrogen fixation has been bolstered by a diverse array of scientific techniques. Still, the origin and extant distribution of nitrogen fixation has been perplexing from a phylogenetic perspective, largely because of factors that confound molecular phylogeny such as sequence divergence, paralogy, and horizontal gene transfer. Here, we make use of 110 publicly available complete genome sequences to understand how the core components of nitrogenase, including NifH, NifD, NifK, NifE, and NifN proteins, have evolved. These genes are universal in nitrogen fixing organisms-typically found within highly conserved operons-and, overall, have remarkably congruent phylogenetic histories. Additional clues to the early origins of this system are available from two distinct clades of nitrogenase paralogs: a group composed of genes essential to photosynthetic pigment biosynthesis and a group of uncharacterized genes present in methanogens and in some photosynthetic bacteria. We explore the complex genetic history of the nitrogenase family, which is replete with gene duplication, recruitment, fusion, and horizontal gene transfer and discuss these events in light of the hypothesized presence of nitrogenase in the last common ancestor of modern organisms, as well as the additional possibility that nitrogen fixation might have evolved later, perhaps in methanogenic archaea, and was subsequently transferred into the bacterial domain.

Converting the NiFeS carbon monoxide dehydrogenase to a hydrogenase and a hydroxylamine reductase.

Substitution of one amino acid for another at the active site of an enzyme usually diminishes or eliminates the activity of the enzyme. In some cases, however, the specificity of the enzyme is changed. In this study, we report that the changing of a metal ligand at the active site of the NiFeS-containing carbon monoxide dehydrogenase (CODH) converts the enzyme to a hydrogenase or a hydroxylamine reductase. CODH with alanine substituted for Cys(531) exhibits substantial uptake hydrogenase activity, and this activity is enhanced by treatment with CO. CODH with valine substituted for His(265) exhibits hydroxylamine reductase activity. Both Cys(531) and His(265) are ligands to the active-site cluster of CODH. Further, CODH with Fe substituted for Ni at the active site acquires hydroxylamine reductase activity.

Carbon monoxide dehydrogenase from Rhodospirillum rubrum produces formate.

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum reversibly catalyzes the oxidation of CO to CO(2) at the active site C-cluster. In this article, the reduction of CO(2) to formate is reported as a slow side reaction catalyzed by both Ni-containing CODH and Ni-deficient CODH. Recently, the structures of R. rubrum CODH and its active site NiFeS cluster (the C-cluster) have been solved. The data in this manuscript describe the formate-producing capability of CODH with or without Ni in the active site.

Electronic, Magnetic, and Redox Properties of [MFe(3)S(4)] Clusters (M = Cd, Cu, Cr) in Pyrococcus furiosus Ferredoxin.

The ground- and excited-state properties of heterometallic [CuFe(3)S(4)](2+,+), [CdFe(3)S(4)](2+,+), and [CrFe(3)S(4)](2+,+) cubane clusters assembled in Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR and variable-temperature/variable-field magnetic circular dichroism (MCD) studies. The results indicate Cd(2+) incorporation into [Fe(3)S(4)](0,-) cluster fragments to yield S = 2 [CdFe(3)S(4)](2+) and S = (5)/(2) [CdFe(3)S(4)](+) clusters and Cu(+) incorporation into [Fe(3)S(4)](+,0) cluster fragments to yield S = (1)/(2) [CuFe(3)S(4)](2+) and S = 2 [CuFe(3)S(4)](+) clusters. This is the first report of the preparation of cubane type [CrFe(3)S(4)](2+,+) clusters, and the combination of EPR and MCD results indicates S = 0 and S = (3)/(2) ground states for the oxidized and reduced forms, respectively. Midpoint potentials for the [CdFe(3)S(4)](2+,+), [CrFe(3)S(4)](2+,+), and [CuFe(3)S(4)](2+,+) couples, E(m) = -470 +/- 15, -440 +/- 10, and +190 +/- 10 mV (vs NHE), respectively, were determined by EPR-monitored redox titrations or direct electrochemistry at a glassy carbon electrode. The trends in redox potential, ground-state spin, and electron delocalization of [MFe(3)S(4)](2+,+) clusters in P. furiosus ferredoxin are discussed as a function of heterometal (M = Cr, Mn, Fe, Co, Ni, Cu, Zn, Cd, and Tl).