The Extended C-Terminal α-Helix of the HypC Chaperone Restricts Recognition of Large Subunit Precursors by the Hyp-Scaffold Machinery during [NiFe]-Hydrogenase Maturation in Escherichia coli.

Members of the HypC protein family are chaperone-like proteins that play a central role in the maturation of [NiFe]-hydrogenases (Hyd). Escherichia coli has a second copy of HypC, called HybG, and, as a component of the HypDEF maturation scaffold, these proteins help synthesize the NiFe-cofactor and guide the scaffold to its designated hydrogenase large subunit precursor. HypC is required to synthesize active Hyd-1 and Hyd-3, while HybG facilitates Hyd-2 and Hyd-1 synthesis. To identify determinants on HypC that allow it to discriminate against Hyd-2, we made amino acid exchanges in 3 variable regions, termed VR1, VR2, and VR3, of HypC, that make it more similar to HybG. Region VR3 includes a HypC-specific C-terminal α-helical extension, and this proved particularly important in preventing the maturation of Hyd-2 by HypC. Truncation of this extension on HypC increased Hyd-2 activity in the absence of HybG, while retaining maturation of Hyd-3 and Hyd-1. Combining this truncation with amino acid exchanges in VR1 and VR2 of HypC negatively affected the synthesis of active Hyd-1. The C-terminus of E. coli HypC is thus a key determinant in hindering Hyd-2 maturation, while VR1 and VR2 appear more important for Hyd-1 matu-ration.

Evidence for an oxygen-sensitive iron-sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2.

Endoprotease-specific C-terminal processing is required to complete the maturation of the large subunit of [NiFe]-hydrogenases. This happens only after synthesis and insertion of the NiFe(CN)(2)CO cofactor by the Hyp maturases has occurred. It is assumed that in the absence of maturation the unprocessed species of the large subunit lacks cofactors. In this study we isolated a variant of the hydrogenase 2 large subunit, HybC, containing a fused C-terminal pentapeptide. The polypeptide could not be processed and was unable to associate with the small subunit to deliver an active enzyme. The His(6)-HybC variant protein isolated was brown and had sub-stoichiometric amounts of an oxygen-sensitive Iron-sulfur cluster, which could be chemically reconstituted to a [4Fe-4S] cluster. This cluster was coordinated by the conserved cysteinyl residues that normally ligate the NiFe(CN)(2)CO cofactor. Our findings provide evidence for temporary promiscuity of cofactor-binding sites.

Structure of hydrogenase maturation protein HypF with reaction intermediates shows two active sites.

[NiFe]-hydrogenases are multimeric proteins. The large subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands is catalyzed by the combined actions of HypF and HypE using carbamoylphosphate as a substrate. We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14 Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.

The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity.

BACKGROUND: Escherichia coli synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H2: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity. RESULTS: Here we report the identification of a new H2: benzyl viologen oxidoreductase enzyme activity in E. coli that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of E. coli mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes. CONCLUSIONS: The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of Escherichia coli have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.