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.

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During the assembly of the Escherichia coli proton-translocating ATPase, the subunits of F1 interact with Fo to increase the proton permeability of the transmembrane proton channel. We tested the involvement of the 8 subunit in this process by partially and completely deleting uncH (8 subunit) (14).Plasmid constructions. The plasmids used in these studies are diagrammed in Fig. 1. Plasmids pWSB35, pRPG28, pRPG23, and pRPG51 have been described previously (2, 10). Plasmid pTN1 was constructed by digesting pWSB35 with NruI and religating. The resultant plasmid, pTN1, coded for the Fo genes uncB, uncE, and uncF and more than half of uncH. Plasmid pTN2 was constructed by digesting pWSB35 with BamHI and religating, resulting in a 617-bp deletion within uncB. Plasmid pEA4 was constructed by using site-directed mutagenesis to insert a Sall site within the second codon of uncH. A 1,499-bp BamHI-EcoRI fragment which carried uncE, uncF, and uncH (c, b, and 8 subunits) was cloned from pRPG23 into M13mpl8. A 36-base synthetic oligonucleotide (5'-GGAGGGAGGGGCT GATGTCGACTTTATTACGGTAGC-3') was annealed to single-stranded DNA isolated from phage produced by cells containing the resultant recombinant phage DNA. This primer contains a sequence complementary to the unc sequence around the ribosome-binding site and initiation codon for uncH, except that it contains a single-base deletion at base 6 (T) of uncH and a single-base substitution at base 9 (A to C) of uncH. We used the Amersham mutagenesis kit (Amersham Corp.) to replace the wild-type uncH sequence with this mutagenized sequence, which resulted in the creation of a Sall site within the second codon of uncH.The BamHI-SalI fragment (containing all of uncE) from the mutagenized plasmid was sequenced in its entirety to ensure the absence of other mutations. This fragment was then cloned into plasmid pTN2, which had been digested with BamHI and Sall, to