Ferredoxin as a Physiological Electron Donor for Carbon Dioxide Fixation to Formate in a Bacterial Carbon Dioxide Reductase

Abstract

Hydrogen-dependent CO 2 reductase (HDCR) is the key enzyme in CO 2 fixation and acetogenesis in some anaerobic, acetogenic bacteria. The enzyme has four subunits, a hydrogenase module HydA2 and a formate dehydrogenase module FdhF that are connected by two small iron–sulfur proteins, HycB3 and HycB4. The enzyme catalyzes the conversion of H 2 + CO 2 to formate and vice versa with the highest rates ever reported. HDCR from Acetobacterium woodii was shown in vitro to also use ferredoxin (Fd) as the electron carrier, a central electron carrier in anaerobes. The same was observed here for the enzyme purified from the thermophile Thermoanaerobacter kivui: the HDCR catalyzed formate production from reduced ferredoxin and CO 2 and vice versa . The enzyme also catalyzed Fd-dependent proton reduction with the production of molecular hydrogen. After deletion of HydA2 and its corresponding subunit HycB4, the purified deletion variants still catalyzed formate-dependent Fd reduction with activities even higher than the wild type enzyme, and likewise, Fd 2– -dependent CO 2 reduction was also stimulated. To determine whether ferredoxin can be utilized by HDCR also in vivo, growth studies were performed with strains producing the different HDCR variants. When HydA2 or HydA2 plus HycB4 were deleted, cells still grew on glucose and still fixed CO 2, although the growth rate and the yield were reduced. Cells did not grow on H 2 + CO 2 but on formate or carbon monoxide. Resting cells without HydA2 did not convert H 2 + CO 2 to acetate. Formate was oxidized by wild type cells mainly to H 2 + CO 2 with little acetate formed, but the deletion variants did not produce H 2 anymore but acetate instead. CO conversion to acetate was similar in the wild type and the deletion strains. These experiments demonstrate that hydrogen is not essential as an electron donor for HDCR in vivo.

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