Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly

Abstract

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN– and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN– and CO ligands on the scaffold complex HypCD.

An in vitro reconstitution system to monitor iron transfer to the active site during the maturation of [NiFe]-hydrogenase

“…Although the use of the in vitro system has yielded significant information concerning the requirements of iron delivery to the active site of [NiFe]-Hyd, the metabolic source of iron as it enters the maturation was not addressed in the current study. The HybG protein (or its homolog HypC) might be involved in recruiting both the iron and CO 2 molecules for further reductive synthesis of the CO ligand by HypD ( 13 , 14 ). In future work, we will investigate whether the iron-bound CO 2 ligand coordinated by HybG or HypC has only catalytic function or it ends up in the catalytic site.…”

“…All strains and plasmids used are listed in Table 4 . To produce the recombinant GD complex, the plasmid pThypDEFC StrepXT ( 14 ) was digested with NdeI and BamH1 releasing the hypC gene. The PCR-amplified hybG gene was then ligated into the NdeI and BamH1 restriction sites of pT-hypDEF to generate the pT-hypDEFG StrepXT plasmid encoding a twin-Strep-tag fused to HybG at the C terminus.…”

“…In a recent study, we proposed a unique redox cascade between the [4Fe–4S] cluster and a conserved disulfide motif of HypD. This provided comprehensive evidence for an electron inventory fit to drive multi–electron redox reactions required for the attachment of the CN − and CO ligands to the iron ion ( 14 ).…”

“…1 ) suggests the following sequence of events: (1) the HybG dimer is proposed to deliver both iron and CO 2 to HypD ( 13 ). Subsequently, the HybG–HypD (GD) complex is formed while a HybG monomer is released ( 14 ). (2) The GD complex is proposed to catalyze an ATP-dependent reduction of CO 2 ( 15 ); however, definite proof for this enzymatic activity is yet to obtain.…”

“…The transfer of each CN − ligand to the iron ion on the GD complex requires the input of 2 electrons ( 17 , 24 ). In addition, 2 electrons are required for CO 2 reduction if CO 2 is the metabolic precursor of CO ( 14 ). HypD must be involved in electron transfer because it is the only maturation enzyme that carries an [4Fe–4S] cluster.…”