ABSTRACTStaphylococcus aureusis the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis in the developed world. The ability ofS. aureusto cause substantial disease in distinct host environments is supported by a flexible metabolism that allows this pathogen to overcome challenges unique to each host organ. One feature of staphylococcal metabolic flexibility is a branched aerobic respiratory chain composed of multiple terminal oxidases. Whereas previous biochemical and spectroscopic studies reported the presence of three different respiratory oxygen reductases (otype,bdtype, andaa3type), the genome contains genes encoding only two respiratory oxygen reductases,cydABandqoxABCD. Previous investigation showed thatcydABandqoxABCDare required to colonize specific host organs, the murine heart and liver, respectively. This work seeks to clarify the relationship between the genetic studies showing the unique roles of thecydABandqoxABCDin virulence and the respiratory reductases reported in the literature. We establish that QoxABCD is anaa3-type menaquinol oxidase but that this enzyme is promiscuous in that it can assemble as abo3-type menaquinol oxidase. However, thebo3form of QoxABCD restricts the carbon sources that can support the growth ofS. aureus. In addition, QoxABCD function is supported by a previously uncharacterized protein, which we have named CtaM, that is conserved in aerobically respiringFirmicutes. In total, these studies establish the heme A biosynthesis pathway inS. aureus, determine that QoxABCD is a typeaa3menaquinol oxidase, and reveal CtaM as a new protein required for typeaa3menaquinol oxidase function in multiple bacterial genera.IMPORTANCEStaphylococcus aureusrelies upon the function of two terminal oxidases, CydAB and QoxABCD, to aerobically respire and colonize distinct host tissues. Previous biochemical studies support the conclusion that a third terminal oxidase is also present. We establish the components of theS. aureuselectron transport chain by determining the heme cofactors that interact with QoxABCD. This insight explains previous observations by revealing that QoxABCD can utilize different heme cofactors and confirms that the electron transport chain ofS. aureusis comprised of two terminal menaquinol oxidases. In addition, a newly identified protein, CtaM, is found to be required for the function of QoxABCD. These results provide a more complete assessment of the molecular mechanisms that support staphylococcal respiration.