Abstract
Background[FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H2. These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated.Principal FindingsWe report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation.SignificanceThe in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.
Highlights
Hydrogenases are subdivided into three classes: [NiFe] hydrogenases, [FeFe] hydrogenases, and [Fe] hydrogenases, each characterized by a unique active site cofactor [1,2,3,4,5]. [NiFe] and [FeFe] hydrogenases catalyze the reversible oxidation of dihydrogen: H2O2H++2e2
HydA1 apohydrogenase was heterologously produced in E. coli in the absence of the maturases and purified using immobilized metal-affinity chromatography (IMAC)
Pooled fractions contained high purity HydA1 based on SDS-polyacrylamide gels visualized with Coomassie stain (Fig. 1A)
Summary
Hydrogenases are subdivided into three classes: [NiFe] hydrogenases, [FeFe] hydrogenases, and [Fe] hydrogenases, each characterized by a unique active site cofactor [1,2,3,4,5]. [NiFe] and [FeFe] hydrogenases catalyze the reversible oxidation of dihydrogen: H2O2H++2e2. Subsequent recombinant co-expression of the C. reinhardtii [FeFe] hydrogenase with C. reinhardtii HydEF and HydG in E. coli enabled production of active hydrogenase [8] Following this discovery, in vitro work with the individual maturases has shed light on their respective roles in the synthesis of the Hcluster cofactor and its insertion into the hydrogenase active site. Various recombinant systems have demonstrated active [FeFe] hydrogenase synthesis, both in vivo [8,13,14,15] and in vitro [12,14,16], and other in vitro metalloenzyme systems have shown improved post-translational activation following incubation of the apoproteins with their respective maturases along with exogenous small molecules [17,18] Despite these advancements, [FeFe] hydrogenase studies have far failed to demonstrate enhanced. This discovery provided a unique opportunity to identify which small molecules play a role in hydrogenase activation and H-cluster biosynthesis
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