Transient-state reduction and steady-state kinetic studies of menaquinol oxidase from Bacillus subtilis, cytochrome aa(3)-600 nm. Spectroscopic characterization of the steady-state species

Cytochrome aa(3)-600 or menaquinol oxidase, from Bacillus subtilis, is a me mber of the hemecopper oxidase family. Cytochrome aa(3)-600 contains cytoch rome a, cytochrome a(3), and Cu-B, and each is coordinated via histidine re sidues to subunit I. Subunit II of cytochrome aa(3)-600 lacks Cu-A, which i s a common feature of the cytochrome c oxidase family members. Anaerobic re duction of cytochrome aa(3)600 by the substrate analogue 2,3-dimethyl-1,4-n aphthoquinone (DMN) resolves two distinct kinetic phases by stopped-flow, s ingle-wavelength spectrometry. Global analysis of time-resolved, multiwavel ength spectra shows that during these distinct phases cytochromes a and a3 are both reduced. Cyanide binding to cytochrome a3 enhances the fast phase rate, which in the presence of cyanide can be assigned to cytochrome a redu ction, whereas cytochrome a(3)-cyanide reduction is slow. The steady-state activity of cytochrome aa3-600 exhibits saturation kinetics as a function o f DMN concentration with a K-m of 300 muM and a maximal turnover of 63.5 s( -1). Global kinetic analysis of steady-state spectra reveals a species that is characteristic of a partially reduced oxygen adduct of cytochrome a(3)- Cu-B, whereas cytochrome a remains oxidized. Electron paramagnetic resonanc e (EPR) spectroscopy of the oxidase in the steady state shows the expected signal from ferricytochrome a, and a new EPR signal at g = 2.01. A model of the catalytic cycle for cytochrome aa(3)-600 proposes initial electron del ivery from DMN to cytochrome a, followed by rapid heme to heme electron tra nsfer, and suggests possible origins of the radical signal in the steady-st ate form of the enzyme.