Datensatz

Zusammenfassung

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000−1000 cm^-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800−1000 cm^-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620−1680 cm^-1) and amide II (ca. 1560−1540 cm^-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600−1200 cm^-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm^-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mäntele, W. (1996) FEBS Lett.385, 53−57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (−0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm^-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm^-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm^-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.