title: FTIR detection of protonation/deprotonation of key carboxyl side chains caused by redox change of the Cu(A)-heme a moiety and ligand dissociation from the heme a3-Cu(B) center of bovine heart cytochrome c oxidase.
abstract: FTIR spectral changes of bovine cytochrome c oxidase (CcO) upon ligand dissociation from heme a(3)() and redox change of the Cu(A)-heme a moiety (Cu(A)Fe(a)()) were investigated. In a photosteady state under CW laser illumination at 590 nm to carbonmonoxy CcO (CcO-CO), the C-O stretching bands due to Fe(a3)()(2+)CO and Cu(B)(1+)CO were identified at 1963 and 2063 cm(-)(1), respectively, for the fully reduced (FR) state [(Cu(A)Fe(a)())(3+)Fe(a3)()(2+)Cu(B)(1+)] and at 1965 and 2061 cm(-)(1) for the mixed valence (MV) state [(Cu(A)Fe(a)())(5+)Fe(a3)()(2+)Cu(B)(1+)] in H(2)O as well as in D(2)O. For the MV state, however, another band due to Cu(B)(1+)CO was found at 2040 cm(-)(1), which was distinct from the alpha/beta conformers in the spectral behaviors, and therefore was assigned to the (Cu(A)Fe(a)())(4+)Fe(a3)()(3+)Cu(B)(1+)CO generated by back electron transfer. The FR-minus-oxidized difference spectrum in the carboxyl stretching region provided two negative bands at 1749 and 1737 cm(-)(1) in H(2)O, which were apparently merged into a single band with a band center at 1741 cm(-)(1) in D(2)O. Comparison of these spectra with those of bacterial enzymes suggests that the 1749 and 1737 cm(-)(1) bands are due to COOH groups of Glu242 and Asp51, respectively. A similar difference spectrum of the carboxyl stretching region was also obtained between (Cu(A)Fe(a)())(3+)Fe(a3)()(2+)Cu(B)(1+)CO and (Cu(A)Fe(a)())(5+)Fe(a3)()(2+)Cu(B)(1+)CO. The results indicate that an oxidation state of the (Cu(A)Fe(a)()) moiety determines the carboxyl stretching spectra. On the other hand, CO-dissociated minus CO-bound difference spectra in the FR state gave rise to a positive and a negative peaks at 1749 and 1741 cm(-)(1), respectively, in H(2)O, but mainly a negative peak at 1735 cm(-)(1) in D(2)O. It was confirmed that the absence of a positive peak is not caused by slow deuteration of protein. The corresponding difference spectrum in the MV state showed a significantly weaker positive peak at 1749 cm(-)(1) and an intense negative peak at 1741 cm(-)(1) (1737 cm(-)(1) in D(2)O). The spectral difference between the FR and MV states is explained satisfactorily by the spectral change induced by the electron back flow upon CO dissociation as described above. Thus, the changes of carboxyl stretching bands induced both by oxidation of (Cu(A)Fe(a)()) and dissociation of CO appear at similar frequencies ( approximately 1749 cm(-)(1)) but are ascribed to different carboxyl side chains.
authors.affiliation: Department of Photoscience, The Graduate University for Advanced Studies, Center for Intergarative Bioscience, Okazaki National Research Institutes, Myodaiji, Okazaki, 444-8585, Japan.
authors.forename: Daichi
authors.initials: D
authors.lastname: Okuno
authors.forename: Tadashi
authors.initials: T
authors.lastname: Iwase
authors.forename: Kyoko
authors.initials: K
authors.lastname: Shinzawa-Itoh
authors.forename: Shinya
authors.initials: S
authors.lastname: Yoshikawa
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
mesh_terms: D000818:Animals; D002417:Cattle; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D017666:Deuterium Oxide / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014867:Water / Q000737:chemistry
chemical_list: D008024:Ligands; D012996:Solutions; D014867:Water; C027728:heme a; D006418:Heme; D003300:Copper; D003576:Electron Transport Complex IV; D017666:Deuterium Oxide