Azorhizobium caulinodans electron-transferring flavoprotein N electrochemically couples pyruvate dehydrogenase complex activity to N-2 fixation

Azorhizobium caulinodans thermolabile point mutants unable to fix N-2 at 42 degreesC were isolated and mapped to three, unlinked loci; from complementation tests, several mutants were assigned to the fixABCX locus. Of these, two independent fixB mutants carried missense substitutions in the product electron-transferring flavoprotein N (ETFN) alpha-subunit. Both thermolabile missense variants Y238H and D229G mapped to the ETF(N)alpha interdomain linker. Unlinked thermostable suppressors of these two fixB missense mutants were identified and mapped to the lpdA gene, encoding dihydrolipoamide dehydrogenase (LpDH), immediately distal to the pdhABC genes, which collectively encode the pyruvate dehydrogenase (PDH) complex. These two suppressor alleles encoded LpDH NAD-bincling domain missense mutants G187S and E210G. Crude cell extracts of these fixB lpdA double mutants showed 60-70% of the wild-type PDH activity; neither fixB lpdA double mutant strain exhibited any growth phenotype at the restrictive or the permissive temperature. The genetic interaction between two combinations of lpdA and fixB missense alleles implies a physical interaction of their respective products, LpDH and ETFN. Presumably, this interaction electrochemically couples LpDH as the electron donor to ETFN as the electron acceptor, allowing PDH complex activity (pyruvate oxidation) to drive soluble electron transport via ETFN to N-2, which acts as the terminal electron acceptor. If so, then, the A. caulinodans PDH complex activity sustains N-2 fixation both as the driving force for oxidative phosphorylation and as the metabolic electron donor.