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

Four Hyp proteins build a scaffold complex upon which the Fe(CN)2 CO 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.

Exchange of a Single Amino Acid Residue in the HybG Chaperone Allows Maturation of All H2-Activating [NiFe]-Hydrogenases in Escherichia coli.

The biosynthesis of the NiFe(CN)2CO organometallic cofactor of [NiFe]-hydrogenase (Hyd) involves several discreet steps, including the synthesis of the Fe(CN)2CO group on a HypD-HypC scaffold complex. HypC has an additional function in transferring the Fe(CN)2CO group to the apo-precursor of the Hyd catalytic subunit. Bacteria that synthesize more than one Hyd enzyme often have additional HypC-type chaperones specific for each precursor. The specificity determinants of this large chaperone family are not understood. Escherichia coli synthesizes two HypC paralogs, HypC and HybG. HypC delivers the Fe(CN)2CO group to pre-HycE, the precursor of the H2-evolving Hyd-3 enzyme, while HybG transfers the group to the pre-HybC of the H2-oxidizing Hyd-2 enzyme. We could show that a conserved histidine residue around the amino acid position 50 in both HypC and HybG, when exchanged for an alanine, resulted in a severe reduction in the activity of its cognate Hyd enzyme. This reduction in enzyme activity proved to be due to the impaired ability of the chaperones to interact with HypD. Surprisingly, and only in the case of the HybG H52A variant, its co-synthesis with HypD improved its interaction with pre-HycE, resulting in the maturation of Hyd-3. This study demonstrates that the conserved histidine residue helps enhance the interaction of the chaperone with HypD, but additionally, and in E. coli only for HybG, acts as a determinant to prevent the inadvertent maturation of the wrong large-subunit precursor. This study identifies a new level of control exerted by a bacterium synthesizing multiple [NiFe]-Hyd to ensure the correct enzyme is matured only under the appropriate physiological conditions.

The iron-sulfur-containing HypC-HypD scaffold complex of the [NiFe]-hydrogenase maturation machinery is an ATPase.

HypD and HypC, or its paralogue HybG in Escherichia coli, form the core of the scaffold complex that synthesizes the Fe(CN)2 CO component of the bimetallic NiFe-cofactor of [NiFe]-hydrogenase. We show here that purified HypC-HypD and HybG-HypD complexes catalyse hydrolysis of ATP to ADP (kcat  â‰… 0.85·s-1 ); the ATPase activity of the individual proteins was between 5- and 10-fold lower than that of the complex. Pre-incubation of HypD with ATP was necessary to restore full activity upon addition of HybG. The conserved Cys41 residue on HypD was essential for full ATPase activity of the complex. Together, our data suggest that HypD undergoes ATP-dependent conformational activation to facilitate complex assembly in preparation for substrate reduction.

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.