Electrochemical and ultraviolet/visible/infrared spectroscopic analysis ofheme a and a(3) redox reactions in the cytochrome c oxidase from Paracoccus denitrificans: Separation of heme a and a(3) contributions and assignmentof vibrational modes

Cytochrome c oxidase from Paracoccus denitrificans was studied with a combi ned electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectrosco pic approach. Global fit analysis of oxidative electrochemical redox titrat ions was used to separate the spectral contributions coupled to heme a and as redox transitions, respectively. Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox compon ents (here heme a and a(3)) at all wavelengths in the UV/vis (900-400 nm) a nd at all wavenumbers in the infrared (1800-1250 cm(-1)) yielded difference spectra for the number of redox potentials selected. With an assumption of two redox components, two spectra for the redox potential at -0.03 +/- 0.0 1 V and 0.22 +/- 0.04 V (quoted vs Ag/AgCl) were obtained. The method used here allows the separation of the heme signals from the electrochemically i nduced visible difference spectra of native cytochrome c oxidase without th e addition of any inhibitors. The separated heme a and as UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5 /6-coordinated heme a/a(3) model compounds presented by Babcock [(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wi ley and Sons, New York]. Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fi ts, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transf er of the cofactors (heme a/a(3), Cu-A, and Cu-B), as well from coupled pro cesses such as proton transfer. A tentative assignment of heme vibrational modes is presented and the assignment of the signals to the reorganization of the polypeptide backbone and to perturbations of single amino acids, in particular Asp, Glu, Arg, or Tyr, is discussed.