Abstract
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.
Highlights
The thermophilic bacterium Bacillus stearothermophilus possesses a caa3-type terminal oxidase, which was previously purified
CO Binding—Carbon monoxide binds to the reduced a3-CuB binuclear center of the caa3-type oxidase from B. stearothermophilus with characteristic heme spectral changes (Fig. 1, panel A)
In order to investigate the kinetic features of the electron entry into B. stearothermophilus caa3, we studied the reduction of the cyanide-bound enzyme; when complexed with cyanide, the a3CuB binuclear center cannot be reduced by using ascorbate and TMPD, as shown for bovine cytochrome c oxidase [12, 20]
Summary
(Received for publication, February 13, 1996, and in revised form, March 28, 1996). Alessandro Giuffre , Emilio D’Itri, Silva Giannini, Maurizio Brunori‡, Trees Ubbink-Kok§, Wil N. The optical spectra of this enzyme are a combination of the absorption features typical of c and a hemes, which makes it suitable for a kinetic investigation using transient optical spectroscopy Both cytochrome c and TMPD oxidase activity were shown to be a property of caa from B. stearothermophilus: the highest catalytic activity (Vmax ϭ 80 sϪ1 at 30 °C) was observed with Saccharomyces cerevisiae cytochrome c as substrate [6]. In this study the caa3-type cytochrome c oxidase from B. stearothermophilus has been fully characterized by time-resolved optical spectroscopy combined with singular value decomposition (SVD) analysis. Kinetic analysis of the optical transitions occurring during the reaction of purified caa with O2, CO, dithionite, and ascorbate plus TMPD (with the fully active and the cyanide-inhibited enzyme) demonstrates that caa3-type is a member of the terminal oxidase superfamily [9]. The results demonstrate some striking similarities between this enzyme and the electrostatic complex between mammalian cytochrome c and cytochrome c oxidase with respect to spectroscopic, kinetic, and equilibrium properties
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