| Abstract
| - The molecular processes concomitant with the redox reactions ofwild-type and mutantcytochrome c oxidase from Paracoccusdenitrificans were analyzed by a combination ofproteinelectrochemistry and Fourier transform infrared (FTIR) differencespectroscopy. Oxidized-minus-reducedFTIR difference spectra in the mid-infrared (4000−1000cm-1) reflecting full or stepwise oxidationandreduction of the respective cofactor(s) were obtained. In the1800−1000 cm-1 range, these FTIRdifferencespectra reflect changes of the polypeptide backbone geometry in theamide I (ca. 1620−1680 cm-1)andamide II (ca. 1560−1540 cm-1) region inresponse to the redox transition of the cofactor(s). Inaddition,several modes in the 1600−1200 cm-1 rangecan be tentatively attributed to heme modes. A peakat1746 cm-1 associated with the oxidized formand a peak at 1734 cm-1 associated with thereduced formwere previously discussed by us as proton transfer between Asp or Gluside chain modes in the courseof the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser,U., Ostermeier, C., Michel, H., andMäntele, W. (1996) FEBS Lett.385, 53−57]. These signals were resolved intoseveral componentsassociated with the oxidation of different cofactors. For astepwise potential titration from the fully reducedstate (−0.5 V) to the fully oxidized state (+0.5 V), a smallcomponent at 1738 cm-1 develops inthepotential range of approximately +0.15 V and disappears at morepositive potentials while the maincomponent at 1746 cm-1 appears in the rangeof approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two differentionizable residues involved in redox-induced proton transfer. The major component at 1746cm-1 is completely lost in the FTIRdifferencespectra of the Glu 278 Gln mutant enzyme. In the spectrum of thesubunit I Glu 278 Asp mutant enzyme,the major component of the discussed difference band is lost. Incontrast, the complete difference signalof the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser,and Asp 399 Asn variants, whichare critical residues in the discussed proton pump channel as suggestedfrom structure and mutagenesisexperiments. On the basis of these difference spectra of mutants,we present further evidence that glutamicacid 278 in subunit I is a crucial residue for the redox reaction.Potential titrations performed simultaneouslyfor the IR and for the UV/VIS indicate that the signal related to Glu278 is coupled to the electron transferto/from heme a; however, additional involvement ofCuB electron transfer cannot be excluded.
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