Functionality of Single-Cysteine Mutants in Subunit III of Rhodobacter sphaeroides Cytochrome c Oxidase

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Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water using ferrocytochrome c and conserves the energy of this reaction by translocating protons across the bacterial or inner-mitochrondrial membrane. COX from Rhodobacter sphaeroides is a four subunit transmembrane protein that serves as a model for the mitochondrial enzyme. Subunit I and II contain the redox centers and proton pathways necessary for redox chemistry and proton translocation. The indispensable role of subunit III is an area still being investigated. This work examines the functionality of three mutant forms of COX - one in which all cysteines have been removed from the enzyme (CA1CS3), and two in which single cysteines are reintroduced into CA1CS3 at specific locals in subunit III (A4C, S187C). The single cysteine mutants provide a means to specifically target thiol-reactive probes to areas of interest in COX subunit III. The A4C mutant allows for a probe to be placed at the mouth of the D-channel - an important proton-conducting pathway necessary for the pumping and redox activities of COX. Bioconjugation of S187C would place a probe on an exterior loop which is thought to undergo redox-linked transient conformational changes. All three mutants were expressed and purified, and their absorbance spectra are identical to wildtype, indicating that the heme active centers are unperturbed. SDS-PAGE gels show that all three mutants retain wildtype subunit composition. The oxygen reduction activity of the mutants are also comparable to wildtype, with values between 1200-1600 e−/s∗mol at pH 7.4. In conclusion, these results indicate that the cysteine-free mutant and two mutants in which single cysteines are reintroduced at non-conserved locations retain wildtype functionality, indicating that cytochrome c oxidase subunit III is a candidate for cysteine scanning-mutagenesis studies utilizing thiol-reactive probes.