S-Adenosyl Methionine, Cysteine, Tyrosine, Iron, and Sulfide Stimulate in Vitro [FeFe] Hydrogenase Activation in a Heterologous E. Coli System

[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 at least three maturation enzymes for its assembly; the biosynthetic pathways and direct biochemical precursors for which have yet to be elucidated. In this work, we identify small molecules that cooperatively stimulate post-translational [FeFe] hydrogenase activation, providing the first evidence for in vitro synthesis of the H-cluster cofactor. The system is comprised of purified hydrogenase apoprotein; heterologous HydE, HydF, and HydG maturases; and exogenous substrates. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with ferrous iron, inorganic sulfide, S-adenosyl methionine, cysteine, tyrosine, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous small molecules. Moreover, we have begun investigating post-translational hydrogenase activation using purified maturases to further study the biosynthesis of the H-cluster cofactor from a defined set of small molecules. The in vitro system allows for direct assessment of hydrogenase maturation, establishing a robust means to characterize biosynthesis of the H­-cluster cofactor and its non-protein moieties.