The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb(3) 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 bc(1) complex and the cbb(3) oxygen reductase is not known. The most likely candidates are the diheme cytochrome c(4) and monoheme cytochrome c(5), which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c(4) and c(5) from V. cholerae have been cloned and expressed heterologously in Escherichia coli. It is shown that reduced cytochrome c(4) is a substrate for the purified cbb(3) oxygen reductase and can support steady state oxygen reductase activity of at least 300 e(-1)/s. In contrast, reduced cytochrome c(5) is not a good substrate for the cbb(3) 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 c(4) 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 c(4). 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 c(4) are 240 and 340 mV (vs standard hydrogen electrode), similar to the electrochemical properties of other c(4)-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c(4)-type cytochrome and a cbb(3) oxygen reductase within the beta- and gamma-proteobacterial clades, suggesting that cytochrome c(4) is the likely natural electron donor to the cbb(3) oxygen reductases within these organisms. These would include the beta-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb(3) oxygen reductases are the only terminal oxidases in their respiratory chains, and the gamma-proteobacterium Pseudomonas stutzeri.