Selecting 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 and hydrogen. 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 in nitrogen-fixing conditions. To investigate how native electron transfer chains (ETCs) can be redirected towards nitrogen fixation, we leveraged the strong selective pressure of nitrogen limitation to isolate a suppressor of R. palustris {Delta}fixC 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 NAD+-dependent Fd:NADPH oxidoreductase (Nfn). We present evidence that AadN plays a role in electron transfer to benzoyl-CoA reductase, the key enzyme involved in anaerobic aromatic compound degradation. Our data support a model where the ETC for anaerobic aromatic compound degradation was re-purposed to support nitrogen fixation in the suppressor strain. ImportanceThere is increasing evidence that protein electron carriers like Fd have evolved to form specific partnerships with select electron donors and acceptors to keep native electron transfer pathways insulated from one another. This makes it challenging to integrate a Fd-dependent pathway like biological nitrogen fixation into non-nitrogen-fixing organisms and provide the high-energy reducing power needed to fix nitrogen. Here we show that amino acid substitutions in an electron donor for anaerobic aromatic compound degradation and a Fd involved in nitrogen fixation enabled electron transfer to nitrogenase. This work provides a model system to understand electron transfer chain specificity and how new electron transfer pathways can be evolved for biotechnologically valuable pathways like nitrogen fixation.