Methionine-80-sulfoxide cytochrome c: preparation, purification and electron-transfer capabilities.
In order to explore the electron-transferring properties of methionine-80-sulfoxide cytochrome c, the pure, chromatographically homogeneous methionine-80-sulfoxide cytochrome c was previously published procedure (Ivanetich, K.M., Bradshaw, J.J. and Kaminsky, L.S. (1976) Biochemistry 15, 1144-1153) was found to produce a mixture of products. In the pure derivative, visible spectroscopy indicates that the 695 nm band indicative of the Met-80-Fe coordination is missing, amino acid analysis indicates that only one methionine is modified to the sulfoxide, and the E0' is found to be 240 mV vs. N.H.E. For succinate cytochrome c reductase activity, the Km for modified cytochrome was about one-ninth that of the native protein, while the maximum turnover number of the reductase with the modified protein was only about 54% of that with native protein. In contrast, the activity with cytochrome oxidase measured polarographically using ascorbate and TMPD under two different buffer/pH conditions, gave Km values that were very similar for both the native and modified cytochromes c, but the maximum turnover numbers of the oxidase with the modified protein were less than 40% of native in either buffer. It is concluded that the Met-80-sulfoxide cytochrome c in the reduced form is able to maintain substantially its heme crevice structure and thus maintain Km values similar to those of native protein. However, the low maximum turnover numbers for oxidase activity with the modified protein in the reduced state indicate that electron transfer itself has been significantly decreased, probably because the parity of acid/base and electrostatic interactions of Met-80 sulfur with the Fe in the two redox states has been disrupted.
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000302:isolation & purification; D004579:Electron Transport; D066298:In Vitro Techniques; D008715:Methionine / Q000031:analogs & derivatives; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000378:metabolism; D013386:Succinates / Q000378:metabolism
D003574:Cytochrome c Group; D013386:Succinates; D008715:Methionine; D001205:Ascorbic Acid