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 17O 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 H217O. To determine whether a channel involving the Mn2+ was used for water exit from the active site, samples incubated in 17O2 were allowed to turn over approximately five times before freezing. The 17O, now in the form of H217O, 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.
Schmidt
Bryan
B
Departments of Biochemistry and Chemistry, Michigan State University, East Lansing, MI 48824, USA.
McCracken
John
J
Ferguson-Miller
Shelagh
S
D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D003576:Electron Transport Complex IV / Q000737:chemistry*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D008345:Manganese; D003576:Electron Transport Complex IV; D008274:Magnesium