HypD Is the Scaffold Protein for Fe-(CN)₂CO Cofactor Assembly in [NiFe]-Hydrogenase Maturation

Abstract

[NiFe]-hydrogenases bind a NiFe-(CN)2CO cofactor in their catalytic large subunit. The iron-sulfur protein HypD and the small accessory protein HypC play a central role in the generation of the CO and CN(-) ligands. Infrared spectroscopy identified signatures on an anaerobically isolated HypCD complex that are reminiscent of those in the hydrogenase active site, suggesting that this complex is the assembly site of the Fe-(CN)2CO moiety of the cofactor prior to its transfer to the hydrogenase large subunit. Here, we report that HypD isolated in the absence of HypC shows infrared bands at 1956 cm(-1), 2072 cm(-1), and 2092 cm(-1) that can be assigned to CO, CN(1), and CN(2), respectively, and which are indistinguishable from those observed for the HypCD complex. HypC could not be isolated with CO or CN(-) ligand contribution. Treatment of HypD with EDTA led to the concomitant loss of Fe and the CO and CN(-) signatures, while oxidation by H2O2 resulted in a positive shift of the CO and CN(-) bands by 35 cm(-1) and 20 cm(-1), respectively, indicative of the ferrous iron as an immediate ligation site for the diatomic ligands. Analysis of HypD amino acid variants identified cysteines 41, 69, and 72 to be essential for maturation of the cofactor. We propose a refined model for the ligation of Fe-(CN)2CO to HypD and the role of HypC in [NiFe]-hydrogenase maturation.

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

“…NiFe(CN) 2 CO biosynthesis requires specific maturation machinery, in which six Hyp proteins (HypA-HypF) play key roles (6,7). Four Hyp proteins (HypC-HypF) are involved in the biosynthesis and incorporation of the Fe(CN) 2 CO group (8)(9)(10)(11)(12)(13)(14)(15). After Fe insertion, HypA and HypB insert the Ni ion into the hydrogenase large subunit (16).…”

Biosynthesis of Salmonella enterica [NiFe]-hydrogenase-5: probing the roles of system-specific accessory proteins

“…In general, assembly of active [NiFe]-hydrogenases is achieved via the products of two different operons [8,9]. The proteins produced from specialised [Ni-Fe] cofactor assembly genes located in a hyp operon are essential for building the active site, including the synthesis of the unusual catalytic centre ligands CO and CN [10][11][12]. A dedicated hyp operon is found in every bacterium that produces [NiFe]-hydrogenases [10][11][12].…”

“…The proteins produced from specialised [Ni-Fe] cofactor assembly genes located in a hyp operon are essential for building the active site, including the synthesis of the unusual catalytic centre ligands CO and CN [10][11][12]. A dedicated hyp operon is found in every bacterium that produces [NiFe]-hydrogenases [10][11][12]. In addition, each individual [NiFe]-hydrogenase also requires further, often system-specific, assembly proteins that are often encoded by the same operon as the enzyme itself ( Figure 1) [9].…”

hypD as a Marker for [NiFe]-Hydrogenases in Microbial Communities of Surface Waters

“…The most promising candidate of all maturation genes is the highly conserved hypD, encoding a protein with a ferredoxin:thioredoxin reductase-like [4Fe-4S] cluster and additional cysteine residues that probably work as a redox cascade. In concert with HypC, it works as a scaffold protein that allows the insertion of the Fe(CN) 2 (CO) in the correct oxidation state into the active site of the [NiFe]-hydrogenases (11)(12)(13)(14)(15)(16)(17).…”