Cloning, sequencing, and mutagenesis of the cytochrome c4 gene from Azotobacter vinelandii: characterization of the mutant strain and a proposed new branch in the respiratory chain.

Azotobacter vinelandii is a free-living, nitrogen-fixing bacterium with a branched electron transport chain terminating with two terminal oxidases, cytochromes d and o. Cytochrome o is thought to receive its electrons from cytochromes c. The gene encoding cytochrome c4 has been cloned and sequenced (termed the cycA locus). The deduced amino acid sequence contains a 20 residue signaling peptide sequence on the N-terminal end. Mutagenesis was performed by inserting a Kmr cassette into the structural gene. The subsequent mutant strains showed reduced amounts of cytochromes c (approximately 60% of wild-type levels) based on difference absorption spectra measurements. Heme staining confirmed the complete loss of cytochrome c4 protein in the mutant strains. These mutants could grow and respire normally, like the wild type, under both diazotrophic or non-diazotrophic conditions. Surprisingly, the cytochrome o terminal oxidase was still turning over in membranes from the cycA mutants as evidenced by substrate-reduced CO difference spectra and inhibition experiments with the use of the cytochrome o inhibitor, chlorpromazine. Still, the levels of oxidation by ascorbate-TMPD were greatly reduced in the cycA mutants. Therefore, it is proposed that cytochrome c4 does not exist in complex with cytochrome o as a multi-component terminal oxidase complex, yet still passes electrons to it in parallel like cytochrome c5, as opposed to in an obligate sequential manner with cytochrome c5. In this pathway the proposed new branch is at the ubiquinone to cytochromes c level.