Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN) 2 CO group during maturation of E. coli [NiFe]-hydrogenase 2.

[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN) 2 CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN) 2 CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN) 2 CO group. Using native mass spectrometry of an anaerobically isolated HybG-HypD complex we show that HybG carries the Fe(CN) 2 CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO 2 -related, covalent modifications that depended on the presence of the N-terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN) 2 CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN) 2 CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.