Selecting a new electron transfer pathway for nitrogen fixation uncovers an electron bifurcating-like enzyme involved in anaerobic aromatic compound degradation

Abstract

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 ΔfixC 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.