Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family.

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)2CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In E. coli, 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 hypABFCDE operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an E. coli hyp mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of E. coli was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O2-sensitive H2-uptake HupLS [NiFe]-hydrogenase from D. mccartyi CBDB1 was also synthesized in an active form in E. coli. Together, these findings indicated that HypC from D. mccartyi CBDB1 exhibits promiscuity in its large subunit interaction in E. coli. Based on these findings, we generated amino acid variants of E. coli HybG capable of partial recovery of Hyd-3-dependent H2 production in a hypC hybG 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.