Abstract
<jats:p>Ferredoxin NADP reductases (FNRs) are ubiquitous flavoenzymes that catalyse the reversible transfer of electrons between iron sulfur ferredoxins and the pyridine nucleotide pool, thereby occupying a central position in diverse redox metabolic pathways including photosynthesis, nitrogen fixation, and detoxification of reactive oxygen species. Although FNR activity was demonstrated in cell extracts of Clostridium pasteurianum more than five decades ago, the gene encoding this activity has remained unidentified. In the present study, a systematic bioinformatic screen of all 3,797 predicted proteins from the C. pasteurianum genome was conducted using conserved FAD and NAD(P) interacting residues from structurally characterised reductases as search templates. This analysis identified a single candidate, AQ984 05830, which is annotated as a sporulation protein but possesses all six predicted cofactor interacting residues. Heterologous expression and cytochrome c reduction assays confirmed ferredoxin dependent reductase activity, with a wild type kcat of 0.007 min−1 a value orders of magnitude lower than those reported for canonical FNRs. A parallel genome wide screen further revealed a repertoire of ferredoxin like carriers, suggesting that C. pasteurianum distributes hydrogen derived electrons among multiple ferredoxins to serve diverse metabolic fates, of which NADP⁺ reduction by CpFNR is one. Alanine scanning mutagenesis of five predicted cofactor interacting residues revealed that K68A and K73A mutations abolished activity, whereas T64A, T185A and S202A mutations improved catalytic efficiency (kcat/Km) for NADH by 14 to 18 folds. AlphaFold structure prediction combined with SwissDock and ClusPro molecular docking simulations placed the FAD binding site centrally between the NAD(P)H and ferredoxin binding domains, consistent with the expected electron relay architecture. Structural analysis of the beneficial mutations suggests that disruption of hydrogen bonds flanking a flexible coil (residues 186 199) propagates conformational effects to the NAD(P)H binding loops, rationalising the improved substrate affinities. These findings expand the known functional diversity of the FNR superfamily and suggest an unrecognised role for redox regulation during endospore formation in C. pasteurianum.</jats:p>