Redox dependent conformational changes in the mixed valence form of the cytochrome c oxidase from p. The reorganization of glutamic acid 278 is coupled to the electron transfer from/to heme a and the binuclear center. denitrificans.
In this work we present the separation of FTIR difference signals induced by electron transfer to/from the redox centers of the cytochrome c oxidase from P. denitrificans and compare electrochemically induced FTIR difference spectra with those induced by CO photolysis. FTIR difference spectra of rebinding of CO to the half reduced (mixed valence) form of the cytochrome c oxidase after photolysis reflect the conformational changes induced by the rebinding of CO and by electron transfer reactions from heme a3 to heme a and further on to CUA. During this process, heme a3 (and CUB) are oxidized, whereas heme a and CuA are reduced. By subtracting these difference spectra from an electrochemically induced FTIR difference spectrum, where all four cofactors are reduced, the contributions for heme a3 (and CuB) could be separated. Correspondingly, the spectral contributions of heme a and CuA have been separated. The comparison of these spectra with the spectra calculated for the hemes on the basis of their redox dependent changes previously published in Hellwig et al., (Biochemistry 38, (1999) 1685-1694) show a high degree of similarity, except for additional signals coupled to the reorganization of the binuclear center upon CO rebinding. The separated spectra clearly show that the signals attributed to Glu278, an amino acid discussed to be crucial for proton pumping, is coupled to electron transfer to/from heme a and the binuclear heme a3-CuB center.
Hellwig
P
P
Institut für Biophysik, Johann-Wolfgang-Goethe-Universität, Frankfurt, Germany. hellwig@biophysik.uni-frankfurt.de
Rost
B
B
Mäntele
W
W
D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry*; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared
C027728:heme a; D018698:Glutamic Acid; D006418:Heme; D003576:Electron Transport Complex IV