New Electron-Transfer Chain to a Flavodiiron Protein\nin <i>Fusobacterium nucleatum</i> Couples Butyryl-CoA Oxidation\nto O<sub>2</sub> Reduction

<i>Fusobacterium nucleatum</i>, a Gram-negative obligate\nanaerobe, is common to the oral microbiota, but the species is known\nto infect other sites of the body where it is associated with a range\nof pathologies. At present, little is known about the mechanisms by\nwhich <i>F. nucleatum</i> mitigates against oxidative and\nnitrosative stress. Inspection of the <i>F. nucleatum</i> subsp. <i>polymorphum</i> ATCC 10953 genome reveals that\nit encodes a flavodiiron protein (FDP; FNP2073) that is known in other\norganisms to reduce NO to N<sub>2</sub>O and/or O<sub>2</sub> to H<sub>2</sub>O. FNP2073 is dicistronic with a gene encoding a multicomponent\nenzyme termed BCR for <u>b</u>utyryl-<u>C</u>oA <u>r</u>eductase. BCR is composed of a butyryl-CoA\ndehydrogenase domain (BCD), the C-terminal domain of the α-subunit\nof the electron-transfer flavoprotein (Etfα), and a rubredoxin\ndomain. We show that BCR and the FDP form an α<sub>4</sub>β<sub>4</sub> heterotetramic complex and use butyryl-CoA to selectively\nreduce O<sub>2</sub> to H<sub>2</sub>O. The FAD associated with the\nEtfα domain (α-FAD) forms red anionic semiquinone (FAD<sup>•–</sup>), whereas the FAD present in the BCD domain\n(δ-FAD) forms the blue-neutral semiquinone (FADH<sup>•</sup>), indicating that both cofactors participate in one-electron transfers.\nThis was confirmed in stopped-flow studies where the reduction of\noxidized BCR with an excess of butyryl-CoA resulted in rapid (<1.6\nms) interflavin electron transfer evidenced by the formation of the\nFAD<sup>•–</sup>. Analysis of bacterial genomes revealed\nthat the dicistron is present in obligate anaerobic gut bacteria considered\nto be beneficial by virtue of their ability to produce butyrate. Thus,\nBCR-FDP may help to maintain anaerobiosis in the colon.