A study on the reaction mechanism of adenosine 5′-phosphosulfate reductase from Thiobacillus thioparus, an iron-sulfur flavoprotein

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Abstract
The reaction mechanism of adenosine 5''-phosphosulfate (APS) reductase (EC 1.8.99.2) from T. thioparus was studied using difference spectrum and stopped-flow techniques. The enzyme-bound FAD was rapidly reduced by sulfite with a 1st order rate constant of 97.1 s-1. The addition of AMP induced further spectral changes in the reduced enzyme which were consistent with the oxidation of FADH2 to the red (anionic) semiquinone.cntdot.FADH and the concomitant reduction of nonheme Fe to the ferrous state. Superoxide dismutase (EC 1.15.1.1) or anaerobiosis inhibited the reduction of cytochrome c by the enzyme only to the extent of 25-35%, indicating the existence of a direct reduction of cytochrome c by the enzyme without involving O2-. The activity of enzyme with cytochrome c was inhibited by increasing the potassium phosphate concentration, the inhibition being more pronounced with horse heart cytochrome c than with Candida krusei cytochrome c.