[FeFe]-hydrogenase maturation: bridging dithiomethylamine ligand assembly during the biosynthesis of the H-cluster

Abstract

[FeFe]-hydrogenases (HydA) demonstrated the highest catalytic competency to synthesize hydrogen gas by reducing protons under physiological conditions and has become a model catalyst in biohydrogen production. In vivo biosynthesis of the active site H-cluster of HydA requires a complex biocatalytic process that involves two radical S- adenosyl-L-methionine enzymes: HydG and HydE, and a [4Fe-4S] cluster binding GTPase: HydF. The maturation of the H-cluster starts with HydG, which cleaves tyrosine to synthesize CO and CN- ligands and provides [Fe II(CO) 2(CN)(kappa 3-cys)]- (synthon) as the product. Synthon is directly delivered to the second radical SAM enzyme HydE that is hypothesized to adenosylate and reduce two equivalents of synthon and subsequently dimerize and form [Fe I 2(mu-SH) 2(CO) 4(CN) 2] 2- ([2Fe] E). The final step in the assembly of the [2Fe] subcluster is DTMA ligand synthesis. In addition to HydG, HydE, and HydF chemistry, the in vitro maturation of HydA was also dependent on E. coli cell extract as a separate component presumed to provide essential components for the assembly of DTMA. We discovered the activating components as H-protein and T-protein of the glycine cleavage system (GCS), serine hydroxymethyltrans-ferase (SHMT), and small molecule substrates: serine and ammonium. Isotope labeling studies and ENDOR spectroscopy analysis revealed that the carbon and nitrogen atoms of DTMA ligand originate from serine and NH 4 +. We hypothesized that the DTMA ligand is assembled by aminomethyl-lipoyl-H-protein (H met) on the HydF-[2Fe] E complex. Consecutively, we demonstrated that HydA can be matured from a chemically synthesized [2Fe] E precursor, bypassing HydG and HydE, via a lysate-free semisynthetic maturation system with GCS components, SHMT, and corresponding substrates. Results emphasized the role of H met as the precursor for DTMA ligand. Refinement to the semisynthetic maturation, which now includes the [Fe-S] cluster carrier protein NfuA and high-CO-affinity variant Mb-H64L, resulted in significantly enhanced hydrogenase activities reaching 940 micromol H 2 min -1 mg - 1 HydA, a level of activity matches with catalytic rates of HydA matured in vivo. Under the refined semisynthetic maturation conditions, we determined that the [4Fe-4S] cluster of HydF is required to mature HydA. Results from site-directed mutagenesis studies on the HydF scaffold suggest that the [4Fe-4S] cluster plays a structural role in the maturation of HydA.