Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants.
The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).
Wang
K
K
Department of Chemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Zhen
Y
Y
Sadoski
R
R
Grinnell
S
S
Geren
L
L
Ferguson-Miller
S
S
Durham
B
B
Millett
F
F
D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009994:Osmolar Concentration; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D014461:Ultracentrifugation
D003574:Cytochrome c Group; D003576:Electron Transport Complex IV