Evolving a New Electron Transfer Pathway for Nitrogen Fixation Uncovers an Electron Bifurcating-Like Enzyme Involved in Anaerobic Aromatic Compound Degradation

Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin (Fd) or flavodoxin (Fld) to reduce dinitrogen gas (N2) to produce ammonia, generating hydrogen gas (H2) as an obligate product of this activity. Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes multiple proteins that can reduce Fd, the FixABCX complex is the only one shown to support nitrogen fixation, and R. palustris Fix- mutants grow poorly under nitrogen- fixing conditions. To investigate how native electron transfer chains (ETCs) can be redirected toward nitrogen fixation, we leveraged the strong selective pressure of nitrogen limitation to isolate a suppressor of an R. palustris DfixC strain that grows under nitrogen- fixing conditions. We found two mutations were required to restore growth under nitrogen-fixing conditions in the absence of functional FixABCX. One mutation was in the gene encoding the primary Fd involved in nitrogen fixation, fer1, and the other mutation was in aadN, which encodes a homolog of NAD1-dependent Fd:NADPH oxidoreductase (Nfn). We present evidence that AadN plays a role in electron transfer to benzoyl coenzyme A reductase, the key enzyme involved in anaerobic aromatic compound degradation. Our data support a model where the ETC for anaerobic aromatic compound degradation was repurposed to support nitrogen fixation in the DfixC suppressor strain.