Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.
Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.
Alleyne
T A
TA
Department of Chemistry, University of Essex, Colchester, U.K.
Wilson
M T
MT
D003574:Cytochrome c Group / Q000031:analogs & derivatives; D045304:Cytochromes c; D003576:Electron Transport Complex IV; D004735:Energy Transfer; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D015032:Zinc
D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; C050095:zinc cytochrome c; D045304:Cytochromes c; D003576:Electron Transport Complex IV; D015032:Zinc