Six Hyp maturation proteins (HypABCDEF) are conserved in micro-organisms that synthesize [NiFe]-hydrogenases (Hyd). Of these, the HypC chaperones interact directly with the apo-form of the catalytically active large subunit of Hyd enzymes and are believed to transfer the Fe(CN)CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In HypC is specifically required for maturation of Hyd-3 while its paralogue, HybG, is specifically required for Hyd-2 maturation; either HypC or HybG can mature Hyd-1. In this study, we demonstrate that the products of the operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O-sensitive H-uptake HupLS [NiFe]-hydrogenase from CBDB1 was also synthesized in an active form in . Together, these findings indicated that HypC from CBDB1 exhibits promiscuity in its large subunit interaction in . Based on these findings, we generated amino acid variants of HybG capable of partial recovery of Hyd-3-dependent H production in a double null mutant. Together, these findings identify amino acid regions in HypC accessory proteins that specify interaction with the large subunits of hydrogenase and demonstrate functional compatibility of Hyp accessory protein machineries.


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