[NiFe]-hydrogenases (Hyd) bind a nickel-iron-based cofactor. The Fe ion of the
cofactor is bound by two cyanide ligands and a single carbon monoxide ligand.
Minimally six accessory proteins (HypA–HypF) are necessary for NiFe(CN)2CO
cofactor biosynthesis in Escherichia coli. It has been shown that the
anaerobically purified HypC–HypD–HypE scaffold complex carries the Fe(CN)2CO
moiety of this cofactor. In the present study, we have purified the HybG–HypDE
complex and used it to successfully reconstitute in vitro active Hyd from E.
coli. HybG is a homologue of HypC that is specifically required for the
maturation of Hyd-2 and also functions in the maturation of Hyd-1 of E. coli.
Maturation of active Hyd-1 and Hyd-2 could be demonstrated in extracts derived
from HybG- and HypD-deficient E. coli strains by adding anaerobically purified
HybG–HypDE complex. In vitro maturation was dependent on ATP,
carbamoylphosphate, nickel and reducing conditions. Hydrogenase maturation was
prevented when the purified HybG–HypDE complex used in the maturation assay
lacked a bound Fe(CN)2CO moiety. These findings demonstrate that it is
possible to isolate incompletely processed intermediates on the maturation
pathway and to use these to activate apo-forms of [NiFe]-hydrogenase large
subunits
