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

Hydrogen-dependent CO2 reductase (HDCR) is the key enzyme in CO2 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 H2 + CO2 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 CO2 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, Fd2–-dependent CO2 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 CO2, although the growth rate and the yield were reduced. Cells did not grow on H2 + CO2 but on formate or carbon monoxide. Resting cells without HydA2 did not convert H2 + CO2 to acetate. Formate was oxidized by wild type cells mainly to H2 + CO2 with little acetate formed, but the deletion variants did not produce H2 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.