A redox‐active HybG‐HypD scaffold complex is required for optimal ATPase activity during [NiFe]‐hydrogenase maturation in Escherichia coli

Abstract

Four Hyp proteins build a scaffold complex upon which the Fe(CN)2CO group of the [NiFe]‐cofactor of hydrogenases (Hyd) is made. Two of these Hyp proteins, the redox‐active, [4Fe‐4S]‐containing HypD protein and the HypC chaperone, form the basis of this scaffold complex. Two different scaffold complexes exist in Escherichia coli, HypCD, and the paralogous HybG‐HypD complex, both of which exhibit ATPase activity. Apart from a Rossmann fold, there is no obvious ATP‐binding site in HypD. The aim of this study, therefore, was to identify amino acid motifs in HypD that are required for the ATPase activity of the HybG‐HypD scaffold complex. Amino acid‐exchange variants in three conserved motifs within HypD were generated. Variants in which individual cysteine residues coordinating the iron–sulfur ([4Fe‐4S]) cluster were exchanged abolished Hyd enzyme activity and reduced ATPase activity but also destabilized the complex. Two conserved cysteine residues, C69 and C72, form part of HypD's Rossmann fold and play a role in HypD's thiol‐disulfide exchange activity. Substitution of these two residues individually with alanine also abolished hydrogenase activity and strongly reduced ATPase activity, particularly the C72A exchange. Residues in a further conserved GFETT motif were exchanged, but neither hydrogenase enzyme activity nor ATPase activity of the isolated HybG‐HypD complexes was significantly affected. Together, our findings identify a strong correlation between the redox activity of HypD, ATPase activity, and the ability of the complex to mature Hyd enzymes. These results further highlight the important role of thiol residues in the HybG‐HypD scaffold complex during [NiFe]‐cofactor biosynthesis.

Evidence that the Isc Iron-Sulfur Cluster Biogenesis Machinery Delivers Iron for [NiFe]-Cofactor Biosynthesis inEscherichia coli

“…The HypD polypeptide in complexes isolated from the iscU single mutant and the erpA-iscA and entC-feoB double-null mutants had an additional, weak polypeptide that migrated slightly faster (∼39 kDa) than the main HypD polypeptide. This additional polypeptide has been observed previously, but with greater intensity, in HypD amino acid variants lacking a complete [4Fe-4S] cluster [22]. Western blot analyses of the same samples confirmed that the dominant polypeptide migrating at ∼39 kDa was HypD (Fig.…”

“…Because HypD has a [4Fe-4S] cluster [11], and due to the fact that the inability to introduce this cofactor into the enzyme results in its destabilisation and degradation [12, 22], it was important to determine whether native HypD levels were affected in the mutants with defective Isc machineries or iron uptake systems. Therefore, the same extracts used to determine Hyd enzyme activities were analysed by western blotting with antiserum raised against HypD (Fig.…”

“…4a). This absorption shoulder is a characteristic feature of HypD within these complexes [11, 22]. The spectrum of the HypCD complex isolated from the Δ sufA mutant, CP1223 carrying plasmid pT-hypDCStrep (Fig.…”

“…These data indicate that the [4Fe-4S] cluster on HypD can presumably be introduced by the Suf machinery when the Isc system is inactivated. The fact that the [4Fe-4S] cluster was present on HypD was demonstrated by UV-vis spectroscopy, by native-MS and was supported by the fact that these isolated complexes retained a native ATPase activity, which is not the case when HypD lacks its [4Fe-4S] cluster [22].…”

“…When required, eluted, pooled protein fractions from the affinity chromatography steps were immediately buffer-exchanged into anaerobic 50 mM Tris-HCl, pH 8, using 5 ml PD-10 columns containing G-25 Sephadex matrix (Cytiva) and then chromatographed on a 1 ml Q-Sepharose Ò fast-flow column (Cytiva) equilibrated with the same buffer. Bound proteins were eluted stepwise using equilibration buffer containing 50, 150, 300 and 500 mM NaCl [22].…”