A discrete water exit pathway in the membrane protein cytochrome c oxidase

By using the non-redox-active Mg2+/Mn2+ site of cytochrome c oxidase as a probe, water access from the outside of the enzyme and water escape from the buried active site were studied. Water movement was time-resolved by monitoring the magnetic interaction of the oxygen isotope O-17 with the Mn2+ by using a rapid freeze-quench-electron spin echo envelope modulation technique. Rapid (msec) access of water from the bulk phase to the Mn2+ was demonstrated by mixing cytochrome c oxidase with (H2O)-O-17. To determine whether a channel involving the Mn2+ was used for water exit from the active site, samples incubated in O-17(2) were allowed to turn over approximately five times before freezing. The O-17, now in the form of (H2O)-O-17, was detected at the Mn2+. The significant broadening of the Mn2+ signal after the limited number of turnovers strongly suggests that the water exits the protein by means of one discrete pathway, not by random diffusion.