Properties of the [NiFe]‐hydrogenase maturation protein HypD

Abstract

A mutational screen of amino acid residues of hydrogenase maturation protein HypD from Escherichia coli disclosed that seven conserved cysteine residues located in three different motifs in HypD are essential. Evidence is presented for potential functions of these motifs in the maturation process.

[NiFe]‐hydrogenase maturation: Isolation of a HypC–HypD complex carrying diatomic CO and CN− ligands

“…It was previously suggested [5,15,17] that a nucleophilic iron acts as the acceptor for the cyanide ligand generated by the HypFE maturases. Here we have demonstrated that the anaerobically purified HypC Strep -HypD complex carries not only two CN À ligands but also coordinates a single CO.…”

“…1B). Two highly conserved motifs (motif 1: C 41 -G-X-H 44 -X-H and motif 2: G-P-G-C 69 -P 70 -V 71 -C 72 -X-X-P 75 ) within HypD have been shown to be essential for hydrogenase maturation [17] and it is conceivable that the two additional Fe ions are coordinated by these motifs.…”

“…Previous studies have shown that the cysteinyl residue at amino acid position 41 in E. coli HypD is essential for maturase activity [9,17] and because the CN À ligand from HypE can be transferred to the HypCD complex [15], it is therefore conceivable that Cys41 UV-Vis absorption spectra of anaerobically purified HypC Strep -HypD complex (full line; 11 mg ml À1 ), HypC Strep -HypD C41A complex (broad dotted line; 9.5 mg ml À1 ), aerobically purified HypC Strep -HypD complex (fine dotted line; 9.0 mg ml À1 ) and anaerobically isolated HypC Strep -HypD complex (11 mg ml À1 ) treated with 5 mM (dotted line) or 10 mM EDTA (full line) were recorded between 300 and 600 nm. In the interest of clarity the HypC Strep -HypD complexes are designated HypCD with the indicated treatment.…”

“…However, as HypD is the only Hyp protein that has a low-potential [4Fe-4S] 2+ cluster [5,15,16], and a twoelectron reduction step is presumably required for the attachment of each of the ligands to the iron, it is unlikely that the CN À and CO groups are attached to one of the Fe ions of the [4Fe-4S] cluster. Instead, it has been proposed that one of two groups of highly conserved cysteinyl residues play an important role in coordinating the iron atom of the Fe(CN) 2 CO cofactor [17]. In particular, Cys41 of HypD has been shown to be important for enzyme function [17].…”

“…Instead, it has been proposed that one of two groups of highly conserved cysteinyl residues play an important role in coordinating the iron atom of the Fe(CN) 2 CO cofactor [17]. In particular, Cys41 of HypD has been shown to be important for enzyme function [17].…”