Membrane bound cytochrome c oxidase couples the movement of electrons and protons in order to achieve both oxygen reduction and proton translocation across the membrane, resulting in the generation of an electrochemical gradient to support ATP synthesis. Proton movement from the inner surface of cytochrome c oxidase to the active site through the "D" and "K" proton uptake pathways has been identified However, the proton release pathway to the exterior of the protein is not well established. A hydrogen bonded network involving a number of water molecules above the hemes has been postulated to be involved in proton release and to possibly have reversibility for the backflow of protons. It is also proposed that proton backflow may be an important process in physiological regulation of energy efficiency.

To observe and quantify proton pumping and proton backflow in reconstituted vesicles containing cytochrome c oxidase (COV), conditions were established for the use of pH sensitive dyes, phenol red on the outside and fluorescent pyranine trapped on the inside, to allow measurement of proton movement on both sides of the membrane under the same conditions. Stopped-flow techniques were used to follow the catalytic reaction in the millisecond time scale, by fluorescence or rapid scanning visible spectroscopy. The conditions for preparing COVs were optimized by using Biobeads for detergent removal rather than dialysis and by purifying COVs that contained his-tagged oxidase purified by Ni-NTA resin. These procedures increased the size and homogeneity of COVs resulting in improved signal/noise for the internal pH measurements. The size of the vesicles was measured by transmission electron microscopy after fixation with osmium tetroxide by a negative staining method. Several mutants involved in the proton uptake pathway and the postulated proton release/backflow pathways were examined to quantify the rate and extent of proton movement and clarify the role of each proton pathway in cytochrome oxidase activity. Micromolar levels of zinc, an oxidase inhibitor, caused strong inhibition of proton backflow, supporting the idea that proton backflow is important for oxidase activity when uptake of protons from the inside is inhibited, as in the presence of a high membrane potential or by site-directed mutations. Issues still remain concerning the quantitative contributions of protons from the inside and the outside to supporting oxidase activity under various conditions.

The region of H93 has been suggested to be involved in proton pumping in the bovine oxidase and is also a candidate for the inhibitory zinc site. Various mutant forms were produced and tested in order to determine whether H93 has a role in proton pumping or backflow in the bacterial oxidase and whether H93 its alteration diminished zinc inhibition. Various H93 mutants show close to normal proton pumping suggesting H93 is not important in the proton pumping of R. sphaeroides oxidase. Zinc inhibition was also observed in these mutants, ruling out the possibility that H93 plays a role in zinc binding. A decreased activity in the controlled state, particularly at high pH, caused an increased RCR for all the H93 mutants. Since the proton supply from the outside is important in the controlled state, the data suggests that H93 may be involved in proton backflow.

Preliminary results from global fitting and kinetic modeling of the complete spectra from the rapid-scanning data, obtained from the stopped-flow kinetic analysis of proton movements, suggests that this method will provide a more powerful approach to quantitative modeling of proton pumping kinetics. This approach may provide a more definitive answer to the role of proton backflow in oxidase activity and a better understanding of the physiological control of cytochrome c oxidase.