Investigation of the electron-transfer properties of cytochrome c oxidase covalently cross-linked to Fe- or Zn-containing cytochrome c.
Complexes of cytochrome c oxidase and cytochrome c (Fe- or Zn-containing) have been prepared by 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-imide (EDC) cross-linking. The site to which the cytochrome c covalently binds has been identified as being the same, or close to, the site occupied by cytochrome c in the electrostatic complex which may be formed between the proteins. Stopped-flow experiments, monitored either at a single wavelength or through a rapid wavelength-scan facility, showed that covalently bound Fe-containing cytochrome c cannot donate electrons to cytochrome a. Free Fe-containing cytochrome c was, however, able to transfer electrons to cytochrome a in covalent complexes containing either Fe- or Zn-containing cytochrome c. Turnover experiments showed that the complexed enzyme remains catalytically competent but with decreased (40-80%) activity. The steady-state levels of reduction of both free cytochrome c and cytochrome a in the covalent complex were higher than found in the control (uncomplexed) enzyme. These results are discussed with reference to the structure of the covalent complex and lead us to conclude that cytochrome a may accept electrons directly from free cytochrome c and that cross-linking impairs the redox properties of the CuA site.
Alleyne
T A
TA
Department of Chemistry and Biological Chemistry, University of Essex, Colchester, U.K.
Wilson
M T
MT
Antonini
G
G
Malatesta
F
F
Vallone
B
B
Sarti
P
P
Brunori
M
M
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D015032:Zinc / Q000378:metabolism*
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D007501:Iron; D003576:Electron Transport Complex IV; D015032:Zinc