Florian P. Rosenbaum, Anja Poehlein, Rolf Daniel, Volker Müller
ABSTRACT Although the Wood‐Ljungdahl pathway, a wide‐spread pathway for CO 2 fixation in anaerobic microorganisms, was elucidated in the thermophilic acetogenic bacterium Moorella thermoacetica , still relatively little is known about the enzymes involved in hydrogen oxidation, CO 2 fixation, energy conservation and the role of quinones and cytochromes. Here, we have used transcriptomics, enzyme assays and genome analyses to identify missing links. NADPH, generated by a [FeFe] hydrogenase, is the reductant for CO 2 reduction to formate, a key reaction in CO 2 fixation. This reaction is slightly endergonic under standard conditions but becomes thermodynamically feasible at high environmental H 2 concentrations. In addition, formate is taken out of equilibrium by a formate dehydrogenase that potentially forms a complex with an energy‐converting hydrogenase (Fdh–Ech), a novel respiratory enzyme in acetogens. Under low H 2 concentrations, the complex can drive the reverse, endergonic reaction. In addition, we postulate a formate cycle involving a periplasmic, cytochrome b ‐containing formate dehydrogenase. A NADH dehydrogenase‐like enzyme that uses reduced ferredoxin instead of NADH to reduce menaquinone is also involved in energy conservation. The data are summarised in a comprehensive metabolic and bioenergetic model of acetogenesis from H 2 + CO 2 and CO in M. thermoacetica .