During biosynthesis of [NiFe]-hydrogenase 2 (Hyd-2) of Escherichia coli, a 15-amino-acid C-terminal peptide is cleaved from the catalytic large subunit precursor, pro-HybC. This peptide is removed only after NiFe(CN) 2 CO cofactor insertion by the Hyp accessory protein machinery has been completed, suggesting that it has a regulatory function during enzyme maturation. We show here that in hyp mutants that fail to synthesize and insert the NiFe cofactor, and therefore retain the peptide, the Tat (twin-arginine translocon) signal peptide on the small subunit HybO is not removed and the subunit is degraded. In a mutant lacking the large subunit, the Tat signal peptide was also not removed from pre-HybO, indicating that the mature large subunit must actively engage the small subunit to elicit Tat transport. We validated the proposed regulatory role of the C-terminal peptide in controlling enzyme assembly by genetically removing it from the precursor of HybC, which allowed assembly and Tat-dependent membrane association of a HybC-HybO heterodimer lacking the NiFe(CN) 2 CO cofactor. Finally, genetic transfer of the C-terminal peptide from pro-HyaB, the large subunit of Hyd-1, onto HybC did not influence its dependence on the accessory protein HybG, a HypC paralog, or the specific protease HybD. This indicates that the C-terminal peptide per se is not required for interaction with the Hyp machinery but rather suggests a role of the peptide in maintaining a conformation of the protein suitable for cofactor insertion. Together, our results demonstrate that the C-terminal peptide on the catalytic subunit controls biosynthesis, assembly, and membrane association of Hyd-2. IMPORTANCE[NiFe]-hydrogenases are multisubunit enzymes with a catalytic subunit containing a NiFe(CN) 2 CO cofactor. Results of previous studies suggested that after synthesis and insertion of the cofactor by the Hyp accessory proteins, this large subunit changes conformation upon proteolytic removal of a short peptide from its C terminus. We show that removal of this peptide is necessary to allow the cleavage of the Tat signal peptide from the small subunit with concomitant membrane association of the heterodimer to occur. Genetic removal of the C-terminal peptide from the large subunit allowed productive interaction with the small subunit and Tat-dependent membrane insertion of a NiFe cofactor-free enzyme. Results based on swapping of C-terminal peptides between hydrogenases suggest that this peptide governs enzyme assembly via a conformational switch. Biosynthesis of complex metal cofactor-containing, multisubunit enzymes requires strict coordination of cofactor biosynthesis, subunit recruitment, and targeting of the protein to its final cellular location. This is particularly important for the numerous redox enzymes present in bacterial systems, many of which are membrane associated or indeed transported across the cytoplasmic membrane (1). Coordination of biosynthesis and assembly is important because cofactors are often highly complex and ma...

[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.

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...

‘Oxygen-tolerant’ [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.

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