The diheme cytochrome c ₄ from Vibrio cholerae is a natural electron donor to the respiratory cbb ₃ oxygen reductase

The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb₃ oxygen reductase. The cbb ₃ oxygen reductase has been previously isolated and characterized; however, the natural mobile electron donor(s) that shuttles electrons between the bc₁ complex and the cbb₃ oxygen reductase is not known. The most likely candidates are the diheme cytochrome c₄ and monoheme cytochrome c₅, which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c₄ and c₅ from V. cholerae have been cloned and expressed heterologously in Escherichia coli. It is shown that reduced cytochrome c₄ is a substrate for the purified cbb₃ oxygen reductase and can support steady state oxygen reductase activity of at least 300 e^-1/s. In contrast, reduced cytochrome c₅ is not a good substrate for the cbb₃ oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c ₄ does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c₄ indicates that the apparent lack of saturation is due to the strong dependence of K_M and V_max on the concentration of oxidized cytochrome c₄. Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c₄ are 240 and 340 mV (vs standard hydrogen electrode), similar to the electrochemical properties of other c₄-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c₄-type cytochrome and a cbb₃ oxygen reductase within the β- and γ-proteobacterial clades, suggesting that cytochrome c₄ is the likely natural electron donor to the cbb₃ oxygen reductases within these organisms. These would include the β-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb₃ oxygen reductases are the only terminal oxidases in their respiratory chains, and the γ-proteobacterium Pseudomonas stutzeri.