Studies on the Composition of the Mitochondrial Sulfite Oxidase System

Abstract

Making use of the difference in oxygen affinity between sulfite oxidase [EC 1. 8. 3. 1] and the mitochondrial sulfite oxidase system, the composition of the sulfite oxidase system of rat and mouse liver mitochondria was studied polarographically.
Cyanide showed a profound inhibition of sulfite oxidation by mitochondria at lower oxygen concentrations, but did not show any inhibition at higher oxygen concentrations. Sonicated mitochondria and also a crude supernatant fraction from the sonicated mitochondria showed relatively high sulfite oxidation activity at higher oxygen concentrations but the activity diminished as the oxygen concentration decreased. The addition of cytochrome c caused a marked increase in the reaction velocity at lower oxygen concentrations. By gel filtration of a concentrate of the crude supernatant, a finely dispersed particulate fraction containing cytochrome oxidase [EC 1. 9. 3. 1] and a soluble fraction containing sulfite oxidase were separated. Both of these fractions and also cytochrome c were necessary for the reconstitution of the system with high affinity for oxygen. A high oxygen affinity system was also reconstituted in the presence of catalase [EC 1. 11. 1. 6] with cytochrome oxidase, cytochrome c, and a more highly purified preparation of sulfite oxidase. Catalase exerted a protective effect on this system against inactivation during catalysis.
Carefully prepared phosphorylation-coupled mitochondria often, but not always, showed respiratory control when sulfite was used as a substrate. The respiratory control rate observed was about the same as that of ascorbate-tetramethyl-p-phenylene diamine (TMPD), a known electron donor system for cytochrome c. Experimental results suggested that the reduction of cytochrome c was the rate-limiting or nearly rate-limiting step in oxidative phosphorylation of the sulfite oxidase system.
These results provide further support and more direct evidence for the recent view that, in intact mitochondria, electrons are transferred from sulfite oxidase to oxygen via cytochrome c and cytochrome oxidase.
Sulfite inhibited the state 3 oxidation of glutamate by mitochondira, but not those of succinate and malate.