Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans: separation of heme a and a3 contributions and assignment of vibrational modes.

Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach. Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and a3 redox transitions, respectively. Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox components (here heme a and a3) at all wavelengths in the UV/vis (900-400 nm) and 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 +/- 0.01 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 induced visible difference spectra of native cytochrome c oxidase without the addition of any inhibitors. The separated heme a and a3 UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5/6-coordinated heme a/a3 model compounds presented by Babcock [(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wiley and Sons, New York]. Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fits, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transfer of the cofactors (heme a/a3, CuA, and CuB), as well from coupled processes 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.

Protonation of Glu L212 following QB- formation in the photosynthetic reaction center of Rhodobacter sphaeroides: evidence from time-resolved infrared spectroscopy.

The protonation events that occur upon QA-QB-->QAQB- electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mäntele, W., Hienerwadel, R., Lenz, F., Riedel, E. J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lenz, F., Nabedryk, E., Breton, J., Kreutz, W., & Mäntele, W. (1992) Biochemistry 31, 5799-5808]. In the mid-infrared region between 1695 and 1780 cm-1, transient signals associated with QA-QB-->QAQB- electron transfer were observed and characterized. The dominant transient absorbance changes are three positive signals at 1732, 1725, and 1706 cm-1 and two negative signals at 1716 and at 1698 cm-1. The 1725 cm-1-signal disappears upon 1H-->2H exchange as expected for an accessible COOH group and is absent in Glu L212 Gln mutant reaction centers. On this basis, we propose an assignment of this signal to the COOH group of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions from ester C = O groups of bacteriopheophytins cannot be ruled out. In native reaction centers at pH 7 and at 4 degrees C, biphasic kinetics of the transient components were observed at most frequencies. The major signal at 1725 cm-1 exhibits a fast kinetic component of t 1/2 = 0.18 ms (25% of the total amplitude) and a slow one of t1/2 = 1 ms (75% of the total amplitude). A global fit analysis of the signals between 1695 and 1780 cm-1 revealed that the spectral distributions of the fast and the slow components are different. Biphasic kinetics with comparable half-times were also observed for the Glu L212 to Gln mutant. The simplest model to explain these results is that the fast phase represents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The difference spectra of the slow component from native reaction centers show that the 1725 cm-1 band corresponds to an absorbance increase and not to a shift of an existing band. The signal is therefore proposed to arise from the protonation of Glu L212. The amplitude of the 1725 cm-1 signal varies distinctly with pH as expected for protonation of a COO- group. With increasing pH, the amplitude of the slow component increases while that of the fast component decreases slightly.(ABSTRACT TRUNCATED AT 400 WORDS)