Sulfite oxidase from chicken liver. Further characterization of the role of carboxyl groups in the reaction with cytochrome c.
The mitochondrial enzyme sulfite oxidase catalyzes the oxidation of cytochrome c by sulfite. The reaction is inhibited when the enzyme is treated with N-cyclohexyl-N'-[2-(N-methylmorpholino)-ethyl]carbodiimide p-toluenesulfonate (CMC). Inhibition follows the conversion of two carboxyl groups to N-acylurea derivatives. The two groups are about equally reactive toward this inhibitor and blocking of either group abolishes electron transfer to cytochrome c. The rate of inactivation is almost the same in the presence of cytochrome c and under conditions where, on average, 89% of the enzyme is bound to cytochrome c. Therefore, the functional groups are not likely to be at the cytochrome c binding site. There are two equal and non-interacting cytochrome c binding sites per sulfite oxidase monomer. The Kd is 7.5 microM at pH 6.0 and low ionic strength. The data are difficult to reconcile with binding of cytochrome c to a cluster of acidic residues in the area of the heme b prosthetic group, as was envisaged for the cytochrome-b5--cytochrome c complex [Salemme, F.R. (1976) J. Mol. Biol. 102, 563-568]. An improved method for the purification of sulfite oxidase from chicken liver, using affinity chromatography on cytochrome c--Sepharose, is described.
Ritzmann
M
M
Department of Biochemistry, University of Zürich, Switzerland.
Bosshard
H R
HR
D000818:Animals; D001665:Binding Sites; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology; D002645:Chickens; D002846:Chromatography, Affinity; D003574:Cytochrome c Group / Q000378:metabolism*; D004579:Electron Transport; D008930:Mitochondria, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D003574:Cytochrome c Group; D003028:CME-Carbodiimide; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors