Abstract
Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O(2) and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O(2)] with a second-order rate constant of k(ox) = 8.7 x 10(4) +/- 0.5 x 10(4) m(-1)s(-1). When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed.
Highlights
Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c
To gain a better understanding of plant sulfite oxidase, we have examined its oxidative half-reaction in some detail
Biphasic transients were seen for the reaction of reduced enzyme with 532 M O2 (Fig. 3, upper transient), with a fast phase occurring with a rate constant of 47.7 Ϯ 0.4 sϪ1 followed by a slower phase with one of 7.5 Ϯ 0.1 sϪ1 (Fig. 3, upper transient)
Summary
Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. Oxidative Half-reaction of A. thaliana Sulfite Oxidase we present results demonstrating effectively quantitative generation of superoxide by plant sulfite oxidase, both in the reaction of reduced enzyme with O2 and during steady-state turnover. In addition to sulfite, superoxide dismutase and cytochrome c were added as appropriate to final concentrations of 200 units/ml and 94.5 M, respectively.
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