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 developed an efficient protocol for the production and isolation of the functional HypCD complex that facilitated detailed spectroscopic investigations. The results obtained by UV/Vis-, electron paramagnetic Resonance (EPR)-, Resonance Raman-, Fourier-transform infrared (FTIR), and Mössbauer spectroscopy provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions. We demonstrate the redox activity of the HypCD complex reporting the interconversion of the [4Fe-4S]2+/+ couple. Additionally, we observed a reversible redox conversion between the [4Fe-4S]2+ and a [3Fe-4S]+ cluster. MicroScale thermophoresis indicated preferable binding between the HypCD complex and its interaction partner HypEF under reducing conditions. Together, these results suggest a redox cascade involving the [4Fe-4S] cluster and a conserved disulfide bond of HypD that may facilitate the synthesis of the [Fe](CN)2CO cofactor precursor on the HypCD scaffold complex.

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

“…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).…”

“…Washed cell pellets were either used immediately or stored at -80 °C. To produce the 57 Fe-labeled GD complex, 57 Fe-HypD and 57 Fe-HybG, the bacteria were grown anaerobically in modified M9 minimal medium exactly as described previously (14).…”

“…Large material and cell debris were removed by centrifugation of the suspension at 15,000 g for 30 min. The cell free extract obtained after centrifugation was used directly for anaerobic protein purification by chromatography on a Strep-Tactin-XT column (IBA Lifesciences, Goettingen), exactly as described (14,20). Wet cell paste containing his-tagged protein was resuspended at a ratio of 1:6 (w/v) in buffer A (50 mM Tris/HCl, pH 8.0, 300 mM NaCl) including 5 µg/ml DNase and protease inhibitors cocktail.…”

“…The assembly of the [Fe](CN) 2 CO moiety (steps 1-3 in Figure 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 complex (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, J o u r n a l P r e -p r o o f definite proof for this enzymatic activity is yet to obtain.…”

“…The transfer of each CNligand to the iron ion on the GD complex requires the input of two electrons (17,24). Additionally, two 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.…”