Purification and properties of sulfite: Cytochrome c oxido-reductase from Thiobacillus novellus

Abstract

• 1.

  1. Sulfite oxidase (sulfite: cytochrome $\text{c}$ oxidoreductase) was purified from Thiobacillus novellus and the properties were studied.

• 2.

  2. Sulfite oxidase did not require AMP for sulfite oxidation and was distinct from adenosine phosphosulfate reductase of Thiobacillus thioparus.

• 3.

  3. The enzyme reduced either ferricyanide or cytochrome $\text{c}$ with sulfite stoichiometrically, reducing 2 moles of the electron acceptor for every mole of sulfite.

• 4.

  4. the pH optimum of enzyme was around 8 with potassium phosphate buffers.

• 5.

  5. No cofactor requirements were demonstrated for the enzyme activity.

• 6.

  6. The enzyme was specific for sulfite as substrate. Thiosulfate, cysteine, GSH, NANO₂ and NH₂OH did not replace sulfite.

• 7.

  7. The $\text{K}{}_{\mathrm{m}}$ for sulfite at pH 8.0 was determined as 4·10⁻⁵ M and 2·10⁻⁵ M with cytochrome $\text{c}$ and ferricyanide as electron acceptors, respectively. At pH 6.5 the $\text{K}{}_{\mathrm{m}}$ for sulfite was 2·10⁻⁶ M with cytochrome $\text{c}$.

• 8.

  8. Various salts and buffers inhibited the enzyme activity. With NaCl the inhibition was found to be competitive with respect to sulfite. The $\text{K}{}_{\mathrm{i}}$ was calculated as 4.5· 10⁻³ M.

• 9.

  9. The enzymes was strongly inhibited by various sulfhydryl inhibitors inlcuding $\text{p-}\text{hydroxymercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$. The inhibition by $\text{p-}\text{hydroxymercuribenzoate}$ was completely reversed by GSH.

• 10.

  10. Methylene blue, NAD⁺, NADP⁺ and O₂ did not replace Fe(CN)₆³⁻ or cytochrome $\text{c}$ as electron acceptor for sulfite oxidase. T. novellus cytochrome $\text{c}$ was reduced by the enzyme and sulfite and subsequently oxidized by T. novellus cytochrome oxidase.

• 11.

  11. Oxidative phosphorylation coupled to sulfite oxidation with a low P/O ratio was demonstrated in cell-free extracts of T. novellus.

• 12.

  12. It is concluded that sulfite oxidase of T. novellus catalyzes the oxidation of sulfite according to the following equation. $\text{SO}{}_3{}^{\mathrm{2-}}\text{+ 2}\text{cyt}\text{c}\text{Fe}{}^{\mathrm{3+}}\text{+}\text{H}{}_2\text{O}\text{→}\text{SO}{}_2{}^{\mathrm{2-}}\text{+ 2}\text{cyt}\text{c}\text{Fe}{}^{\mathrm{2-}}\text{+ 2}\text{H}{}^{\mathrm{+}}$