The caa(3) terminal oxidase of Bacillus stearothermophilus - Transient spectroscopy of electron transfer and ligand binding

Abstract

The thermophilic bacterium Bacillus stearothermophilus possesses a caa(3)-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R. I. HL, and Konings, W. N. (1989) Ear. J. Biochem. 178, 763-770). We have carried out extensive kinetic experiments on the purified enzyme by stopped-flow time-resolved optical spectroscopy combined with singular value decomposition analysis. The results indicate a striking similarity of behavior between this enzyme and the electrostatic complex between mammalian cytochrome c and cytochrome c oxidase. CO binding to fully reduced caa(3) occurs with a second order rate constant (k = 7.8 x 10(4) M(-1) S-1) and an activation energy (E* = 6.1 kcal mol(-1)) similar to those reported for beef heart cytochrome c oxidase. Dithionite reduces cytochrome a with bimolecular kinetics, while cytochrome a(3) (and Cu-B) is reduced via intramolecular electron transfer. When the fully reduced enzyme is mixed with O-2, cytochrome a(3), and cytochrome c are rapidly oxidized, whereas cytochrome a remains largely reduced in the first few milliseconds. When cyanide-bound caa(3) is mixed with ascorbate plus TMPD, cytochrome c and cytochrome a are synchronously reduced; the value of the second order rate constant (k = 3 x 10(5) M(-1) S-1 at 30 degrees C) suggests that cytochrome c is the electron entry site. Steady-state experiments indicate that cytochrome a has a redox potential higher than cytochrome c. The data from the reaction with O-2 reveal a remarkable similarity in the kinetic, equilibrium, and optical properties of caa(3) and the electrostatic complex cytochrome c/cytochrome c oxidase