The oxidoreductase activity of Rnf balances redox cofactors during fermentation of glucose to propionate in Prevotella

Propionate is a microbial metabolite formed in the gastrointestinal tract, and it affects host physiology as a source of energy and signaling molecule. Despite the importance of propionate, the biochemical pathways responsible for its formation are not clear in all microbes. For the succinate pathway used during fermentation, a key enzyme appears to be missing—one that oxidizes ferredoxin and reduces NAD. Here we show that Rnf [ferredoxin—NAD(+) oxidoreductase (Na(+)-transporting)] is this key enzyme in two abundant bacteria of the rumen (Prevotella brevis and Prevotella ruminicola). We found these bacteria form propionate, succinate, and acetate with the classic succinate pathway. Without ferredoxin:NAD(+) oxidoreductase, redox cofactors would be unbalanced; it would produce almost equal excess amounts of reduced ferredoxin and oxidized NAD. By combining growth experiments, genomics, proteomics, and enzyme assays, we point to the possibility that these bacteria solve this problem by oxidizing ferredoxin and reducing NAD with Rnf [ferredoxin—NAD(+) oxidoreductase (Na(+)-transporting)]. Genomic and phenotypic data suggest many bacteria may use Rnf similarly. This work shows the ferredoxin:NAD(+) oxidoreductase activity of Rnf is important to propionate formation in Prevotella species and other bacteria from the environment, and it provides fundamental knowledge for manipulating fermentative propionate production.