A conserved aspartate residue in [4Fe-4S]-containing HypD is required for [NiFe]-cofactor biosynthesis and for efficient interaction of the HypCD scaffold complex with HypE.

Six Hyp (A through F) proteins synthesize the NiFe(CN)2CO cofactor found in all [NiFe]-hydrogenases. The Fe(CN)2CO moiety of this cofactor is assembled on a separate scaffold complex comprising HypC and HypD. HypE and HypF generate the cyanide ligands from carbamoyl phosphate by converting the carbamoyl moiety to a thiocyanate associated with HypE's C-terminal cysteine residue, within a conserved 'PRIC' motif. Here, we identify amino acid residue D98 in the central cleft of HypD to be required for biosynthesis of the Fe(CN)2CO moiety and for optimal interaction of HypD with HypE. Construction of a D98A amino acid variant of HypD caused near-complete loss of hydrogenase activity in anaerobically grown Escherichia coli cells, while exchange of the structurally proximal, but non-conserved, residue S356 on HypD, did not. Native mass spectrometric analysis of the anaerobically purified HypC-HypDD98A scaffold complex revealed only a low amount of the bound Fe(CN)2CO group. Western blotting experiments revealed that purified scaffold complexes between either HypC or HybG (a paralogue of HypC) with HypD-D98A showed a strongly impaired interaction with HypE. Examination of the HypCDE complex crystal structure from Thermococcus kodakarensis revealed that D98 of HypD lies within a cleft through which the C-terminus of HypE can access the bound iron ion on HypCD. Alphafold3 predictions suggest that the D98 residue interacts with the arginine residue of the 'PRIC' motif at the C-terminus of HypE to position the modified terminal cysteine residue precisely for delivery of cyanide to the iron ion associated with the HypCD complex.