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 KM and Vmax 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.