Tyrosine, cysteine, and S-adenosyl methionine stimulate in vitro [FeFe] hydrogenase activation.
BACKGROUND
[FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H(2). 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.


RESULTS
We 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.


CONCLUSIONS
The 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.
Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.
Jon M
JM
Kuchenreuther
James A
JA
Stapleton
James R
JR
Swartz
D001426:Bacterial Proteins / Q000737:chemistry; D020134:Catalytic Domain; D002474:Cell-Free System; D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008956:Models, Chemical; D012436:S-Adenosylmethionine / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D014443:Tyrosine; D012436:S-Adenosylmethionine; D006859:Hydrogen; D007501:Iron; D006864:Hydrogenase; D003545:Cysteine