The caa3 terminal oxidase of Bacillus stearothermophilus. Transient spectroscopy of electron transfer and ligand binding.
The thermophilic bacterium Bacillus stearothermophilus possesses a caa3-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R. I. R., and Konings, W. N. (1989) Eur. 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 caa3 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 a3 (and CuB) is reduced via intramolecular electron transfer. When the fully reduced enzyme is mixed with O2, cytochrome a3, and cytochrome c are rapidly oxidized, whereas cytochrome a remains largely reduced in the first few milliseconds. When cyanide-bound caa3 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 O2 reveal a remarkable similarity in the kinetic, equilibrium, and optical properties of caa3 and the electrostatic complex cytochrome c/cytochrome c oxidase.
Giuffrè
A
A
Department of Biochemical Sciences and CNR Center of Molecular Biology, University of Rome "La Sapienza," 00185 Rome, Italy.
D'Itri
E
E
Giannini
S
S
Brunori
M
M
Ubbink-Kok
T
T
Konings
W N
WN
Antonini
G
G
D000818:Animals; D001205:Ascorbic Acid; D002151:Calorimetry; D002248:Carbon Monoxide / Q000378:metabolism; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D004227:Dithionite / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000494:pharmacology; D013053:Spectrophotometry / Q000379:methods; D013772:Tetramethylphenylenediamine; D013997:Time Factors
D003574:Cytochrome c Group; D004227:Dithionite; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV; D011190:Potassium Cyanide; D013772:Tetramethylphenylenediamine; D001205:Ascorbic Acid