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) 2 CO 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 O 2 -sensitive H 2 -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 H 2 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.
INTRODUCTION[NiFe]-hydrogenases are widespread amongst archaeal and bacterial species (Vignais & Billoud, 2007). These enzymes can either oxidize H 2 to generate a chemiosmotic proton gradient via a membrane-based electron transfer chain, as well as provide a source of reducing power, or dissipate accumulated intracellular reductant by reducing protons to produce H 2 ; some perform both functions under certain physiological conditions (Lubitz et al., 2014;Pinske et al., 2015;Vignais & Billoud, 2007 (Ogata et al., 2015). The biosynthesis of this cofactor is complicated, requiring the combined activities of six Hyp accessory proteins, whose functions include synthesis and insertion of an Fe(CN) 2 CO moiety into the apo-catalytic subunit followed by introduction of the nickel ion (Böck et al., 2006;Forzi & Sawers, 2007). Subsequent to successful cofactor insertion, a C-terminal peptide present on the large subunit of most [NiFe]-hydrogenases is cleaved by a hydrogenase-specific protease and further assembly of the enzyme can then be completed (Böck et al., 2006;Pinske & Sawers, 2014).The Hyp proteins include HypA and the GTPase HypB, which, together with the peptidyl-prolyl cis/trans isomerase SlyD (Zhang et al., 2005), deliver the nickel ion; HypC, which is a small iron-and CO 2 -binding protein (Soboh et al., 2013); the FeS cluster protein HypD, which acts as a scaffold for assembl...
