The kinetic behavior of chicken liver sulfite oxidase

A comprehensive kinetic study of sulfite oxidase has been undertaken over the pH range 6.0-10.0, including conventional steady-state work as well as rapid kinetic studies of both the reaction of oxidized enzyme with sulfite and reduced enzyme with cytochrome c (III). A comparison of the pH dependence of k(cat), k(red), and k(ox) indicates that k(red) is principally rate limiting above pH 7, but that below this pH the pK dependence of k(cat) is influenced by that of k(ox). The pH independence of k(red) is consistent with our previous proposal concerning the reaction mechanism, in which attack of the substrate lone pair of electrons on a Mo(VI)O-2 unit initiates the catalytic sequence. The pH dependence of k(red)/K-d(sulfite) indicates that a group on the enzyme having a pK(a) of similar to 9.3 must be deprotonated for effective reaction of oxidized enzyme with sulfite, possibly Tyr 322, which from the crystal structure of the enzyme constitutes part of the substrate binding site. There is no evidence for the HSO3-/SO32- pK(a) of similar to 7 in the pH profile for k(red)/K-d(sulfite), suggesting that enzyme is able to oxidize the two equally well. By contrast, k(cat)/K-m(sulfite) and k(red)/K-d(sulfite) exhibit distinct pH dependence (the former is bell-shaped, the latter sigmoidal), again consistent with the oxidative half-reaction contributing to the kinetic barrier to catalysis at low pH. The pH dependence of k(cat)/K-m((cyt c)) (reflecting the second-order rate of reaction of free enzyme with free cytochrome) is bell-shaped and closely resembles that of k(ox)/K-d((cyt c)), reflecting the importance of the oxidative half-reaction in the low substrate concentration regime. The pH profile for k(ox)K(d)((cyt c)) indicates that two groups with a pK(a) of similar to 8 are involved in the reaction of free reduced enzyme with cytochrome c, one of which must be deprotonated and the other protonated. These results are consistent with the known electrostatic nature of the interaction of cytochrome c with its physiological partners.