Haase, A.; Arlt, C.; Sinz, A.; Sawers, G.

[NiFe]-hydrogenases have a bimetallic NiFe(CN)2CO cofactor in their large, catalytic subunit. The 136 Da Fe(CN)2CO group of this cofactor is assembled on a distinct HypC-HypD scaffold complex prior to delivery to the apo-catalytic subunit, but the intracellular source of the iron ion is unresolved. Native mass spectrometric (native MS) analysis of HypCD complexes defined the [4Fe-4S] cluster associated with HypD and identified +26 - 28 Da and +136 Da modifications specifically associated with HypC. A HypCC2A variant dissociated from its complex with native HypD lacked all modifications. HypC dissociated from HypCD complexes isolated from Escherichia coli strains deleted for the iscS or iscU genes, encoding core components of the Isc iron-sulfur cluster biogenesis machinery, specifically lacked the +136 Da modification; however, it was retained on HypC isolated from suf mutants. The presence or absence of the +136 Da modification on the HypCD complex correlated with the hydrogenase enzyme activity profiles of the respective mutant strains. Notably, the [4Fe-4S] cluster on HypD was identified in all HypCD complexes analyzed. These results suggest that the iron of the Fe(CN)2CO group on HypCD derives from the Isc machinery, while either the Isc or the Suf machinery can deliver the [4Fe-4S] cluster to HypD.