# 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

## TL;DR

A specific aspartate residue in HypD is crucial for hydrogenase cofactor assembly and interaction with HypE.

## Contribution

Identifies HypD's D98 as essential for [NiFe]-cofactor biosynthesis and HypCD-HypE interaction.

## Key findings

- D98 in HypD is required for Fe(CN)2CO assembly and interaction with HypE.
- D98A mutation in HypD leads to loss of hydrogenase activity in E. coli.
- D98 interacts with HypE's PRIC motif to position cyanide ligands.

## Abstract

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.

Graphical AbstractThe C-terminal cysteine residue of HypE is on a flexible peptide sequence bearing a highly conserved ‚PRIC’ motif. Postioning of the thiocyanate modification on the cysteine close to the Fe ion coordinated by the HypCD complex is facilitated by an electrostatic interaction between the D98-carboxylate of HypD and R334 of HypE’s ‚PRIC‘ motif.

The C-terminal cysteine residue of HypE is on a flexible peptide sequence bearing a highly conserved ‚PRIC’ motif. Postioning of the thiocyanate modification on the cysteine close to the Fe ion coordinated by the HypCD complex is facilitated by an electrostatic interaction between the D98-carboxylate of HypD and R334 of HypE’s ‚PRIC‘ motif.