Although Neisseria gonorrhoeae is a prolific source of eight c-type cytochromes, little is known about how its electron transfer pathways to oxygen are organized. In this study, the roles in the respiratory chain to oxygen of cytochromes c 2, c 4, and c 5, encoded by the genes cccA, cycA, and cycB, respectively, have been investigated. Single mutations in genes for either cytochrome c 4 or c 5 resulted in an increased sensitivity to growth inhibition by excess oxygen and small decreases in the respiratory capacity of the parent, which were complemented by the chromosomal integration of an ectopic, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of the cycA or cycB gene. In contrast, a cccA mutant reduced oxygen slightly more rapidly than the parent, suggesting that cccA is expressed but cytochrome c 2 is not involved in electron transfer to cytochrome oxidase. The deletion of cccA increased the sensitivity of the cycB mutant to excess oxygen but decreased the sensitivity of the cycA mutant. Despite many attempts, a double mutant defective in both cytochromes c 4 and c 5 could not be isolated. However, a strain with the ectopically encoded, IPTG-inducible cycB gene with deletions in both cycA and cycB was constructed: the growth and survival of this strain were dependent upon the addition of IPTG, so gonococcal survival is dependent upon the synthesis of either cytochrome c 4 or c 5. These results define the gonococcal electron transfer chain to oxygen in which cytochromes c 4 and c 5, but not cytochrome c 2, provide alternative pathways for electron transfer from the cytochrome bc 1 complex to the terminal oxidase cytochrome cbb 3.

The CcoP subunit of cytochrome oxidase cbb(3) of Neisseria gonorrhoeae is predicted to include a C-terminal extension in which there is a C-A-A-C-H- motif typical of heme attachment sites in c-type cytochromes. Substitutions of key cysteine and histidine residues of this motif resulted in mutants that grew normally in oxygen-sufficient cultures and reduced oxygen at the same rate as the parent strain. In contrast, after oxygen-limited growth in the presence of nitrite, rates of nitrite reduction were significantly lower than those of the parent, consistent with a role for this third heme-binding domain in electron transfer to the nitrite reductase, AniA, located in the outer membrane. As the mutants were still able to reduce nitrite at approximately 65% of the rate of the parent, there are multiple pathways in the gonococcus for electron transfer to AniA. On the basis of sequence similarity between the C-terminal extension of CcoP and cytochrome c(5), it is proposed that cytochrome c(5) might also transfer electrons across the periplasm from the cytochrome bc(1) complex in the cytoplasmic membrane to AniA in the outer membrane. This is the first example of a cytochrome oxidase component that plays a physiologically significant role in nitrite reduction.

Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c 2 , accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c 4 and c 5 transfer electrons to cytochrome oxidase cbb 3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb 3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c 4 or c 5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (⌬c 2 ), cycA (⌬c 4 ), cycB (⌬c 5 ), and ccoP (ccoP-C368A), we demonstrate that cytochrome c 2 is required for electron transfer from cytochrome c 4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c 5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c 2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c 2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c 2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.