A bacterial electron-bifurcating hydrogenase.

Area of Science:

• Biochemistry
• Microbiology
• Bioenergetics

Background:

• The Wood-Ljungdahl pathway is a candidate for early life's energy metabolism, fixing CO2 using hydrogen.
• Acetobacterium woodii employs an ancient pathway with a single ion potential generation site: the ferredoxin-fueled Rnf complex.
• The endergonic nature of hydrogen-based ferredoxin reduction in this pathway has been a long-standing puzzle.

Purpose of the Study:

• To investigate the mechanism by which Acetobacterium woodii overcomes the energy barrier for hydrogen-based ferredoxin reduction.
• To characterize the purified multimeric [FeFe]-hydrogenase (HydABCD) from A. woodii.

Main Methods:

• Purification of a multimeric [FeFe]-hydrogenase (HydABCD) from A. woodii.
• Enzymatic assays to determine the catalytic activity of the hydrogenase, including ferredoxin and NAD(+) reduction.
• Spectroscopic analyses to elucidate the coupling and stoichiometry of the reactions.

Main Results:

• The purified [FeFe]-hydrogenase (HydABCD) contains multiple iron-sulfur clusters.
• The enzyme catalyzes hydrogen-based ferredoxin reduction, but only in the presence of NAD(+).
• NAD(+) reduction is coupled to ferredoxin reduction in a 1:1 stoichiometry, requiring flavin and utilizing electron bifurcation.

Conclusions:

• The multimeric hydrogenase of A. woodii functions as a soluble energy-converting enzyme.
• Electron bifurcation is employed to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD(+) reduction.
• This mechanism provides insight into ancient bioenergetic strategies and the evolution of the Wood-Ljungdahl pathway.