Protolytic reactions on reduction of cytochrome c oxidase studied by ATR-FTIR spectroscopy.
Reduction of cytochrome c oxidase is coupled to proton uptake, and the reduced-minus-oxidized FTIR spectrum should include signatures of protonation of protolytic centers. The major part of the spectrum shows only small differences between acidic and alkaline conditions, which is consistent with the rather weak pH dependence of the proton uptake stoichiometry. Here we aim at revealing redox state-dependent protonatable sites and present a comprehensive investigation over a wide pH range. The reduced-minus-oxidized transition of cytochrome c oxidase from Paracoccus denitrificans was studied by means of Fourier transform infrared spectroscopy in the pH range 5.2-9.5. Effects of pH were analyzed as the difference between reduced-minus-oxidized FTIR spectra at different pH values. Two pH-dependent processes with apparent pKa values of 6.6 and 8.4 and Hill coefficients 0.9 and 0.1, respectively, were found by this methodology. A sharp OH band appears in the IR "water region" on reduction of the enzyme, independent of pH in the range 6.5-9.0, and downshifted by approximately 940 cm-1 on changing the solvent to D2O and by 10 cm-1 on H216O/H218O isotope exchange. This feature of an asymmetric water molecule may belong to water that is produced in the binuclear center upon reduction or to a structured water molecule that loses a hydrogen bond.
Gorbikova
Elena A
EA
Program for Structural Biology and Biophysics, Institute of Biotechnology, PB 65 (Viikinkaari 1), University of Helsinki, Helsinki FIN-00014, Finland. elena.gorbikova@helsinki.fi
Belevich
Nikolai P
NP
Wikström
Mårten
M
Verkhovsky
Michael I
MI
D041961:Deuterium Exchange Measurement; D004563:Electrochemistry / Q000379:methods; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010103:Oxygen Isotopes; D010231:Paracoccus denitrificans / Q000201:enzymology; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D010103:Oxygen Isotopes; D011522:Protons; D003576:Electron Transport Complex IV; D010100:Oxygen