Abstract
Maturation of [NiFe]-hydrogenase requires the insertion of iron, cyanide and carbon monoxide, followed by nickel, to the catalytic core of the enzyme. Hydrogenase maturation factor HypB is a metal-binding GTPase that is essential for the nickel delivery to the hydrogenase. Here we report the crystal structure of Archeoglobus fulgidus HypB (AfHypB) in apo-form. We showed that AfHypB recognizes guanine nucleotide using Asp-194 on the G5 loop despite having a non-canonical NKxA G4-motif. Structural comparison with the GTPγS-bound Methanocaldococcus jannaschii HypB identifies conformational changes in the switch I region, which bring an invariant Asp-72 to form an intermolecular salt-bridge with another invariant residue Lys-148 upon GTP binding. Substitution of K148A abolished GTP-dependent dimerization of AfHypB, but had no significant effect on the guanine nucleotide binding and on the intrinsic GTPase activity. In vivo complementation study in Escherichia coli showed that the invariant lysine residue is required for in vivo maturation of hydrogenase. Taken together, our results suggest that GTP-dependent dimerization of HypB is essential for hydrogenase maturation. It is likely that a nickel ion is loaded to an extra metal binding site at the dimeric interface of GTP-bound HypB and transferred to the hydrogenase upon GTP hydrolysis.
Highlights
Hydrogenases catalyze the inter-conversion of molecular hydrogen into protons and electrons
Previous study showed that GTPc-S-bound HypB from M. jannaschii (MjHypB) exists as a dimer in the crystal structure [27]
GTP-bound E. coli HypB (EcHypB) has a smaller elution volume than its GDP-bound state in size exclusion chromatography, suggesting that HypB has a tendency to dimerize in the presence of GTP
Summary
Hydrogenases catalyze the inter-conversion of molecular hydrogen into protons and electrons. [NiFe]-hydrogenase, the most widely distributed hydrogenase, which contains a nickel and an iron ions coordinated by a network of thiol ligands from cysteine residues in the catalytic core of the large subunit. The Fe ion is chelated by two CN molecules and one CN molecule [1] Formation of this complex catalytic core in the large subunit of [NiFe]-hydrogenase requires accessory proteins encoded by genes designated hypA to hypF in the hyp operon. Synthesized CN ligands are transferred to HypC and HypD to form a Fe(CN) complex, which is delivered to the hydrogenase large subunit precursor [8]. After delivery of the Fe(CN)2CO complex to the large subunit precursor, nickel is transferred to the precursor with aid of protein HypA and HypB. The last step of catalytic core assembly involves protease cleavage, where an isozyme-specific protease cleaves the C-terminal tail of the large subunit [12]
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