ATP-induced spectral changes in cytochrome c oxidase. A kinetic investigation.
Mixing ATP with soluble oxidized cytochrome c oxidase induces a spectral perturbation in the Soret region of the enzyme. This spectral perturbation is observed at ATP concentrations similar to those found to modulate the catalytic activity of cytochrome c oxidase [Malatesta, Antonini, Sarti & Brunori (1987) Biochem. J. 248, 161-165]. The process is reversible and corresponds to a simple binding with Kd = 0.2 mM at 25 degrees C. The absorbance change follows a first-order time course, and analysis of the ATP-concentration-dependence indicates the presence of a rate-limiting monomolecular step that governs the process. From the temperature-dependence of this process, studied at saturating concentrations of ATP, an activation energy of 44 kJ/mol (10.6 kcal/mol) was measured. The spectral perturbation also occurs when cytochrome c oxidase is reconstituted into artificial phospholipid vesicles, with equilibria and kinetics similar to those observed with the soluble enzyme. Mixing ATP with soluble oxidized cyanide-bound cytochrome c oxidase induces a different spectral perturbation, and the apparent affinity of ATP for the enzyme is substantially increased. There is no absolute specificity for ATP, because EGTA, inositol hexakisphosphate, sulphate and phosphate are all able to induce an identical spectral perturbation with the same kinetics, although the value of the apparent Kd is different for the various anions. The presence of Mg2+ ions decreases, in a saturation-dependent fashion, the apparent affinity of cytochrome c oxidase for ATP. The absorbance change can be correlated to the displacement of the Ca2+ bound to cytochrome c oxidase.
Antonini
G
G
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy.
Malatesta
F
F
Sarti
P
P
Vallone
B
B
Brunori
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D013053:Spectrophotometry
D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV; D008274:Magnesium