Alkylation of cytochromes c II. Carboxymethylation of beef and human cytochromes c in the oxidized and reduced forms

Summary Cytochrome c both from beef and human heart was treated with mono-iodoacetic acid at pH 5–6 under specific conditions in the oxidized and the reduced forms. In the oxidized form, carboxymethylation occurred at methionine-65, methionine-80 and histidine-33 in beef cytochrome c and, in addition, at methionine-12 in human cytochrome c . When all of the forms of cytochrome c were carboxymethylated, the oxidase activity and other enzymic properties of cytochrome c were lost. In contrast, the reduced forms of cytochrome c were carboxymethylated at methionine-65 and histidine-33 in beef cytochrome c and in addition at methionine-12 in human cytochrome c , but not at methionine-80 in either case. The carboxymethylation of these reduced forms of cytochrome c had little or no effect on the enzymic behaviour (oxidase activity), absorption spectrum or the extent of reduction by ascorbate. These results suggest that the conformational change on oxidation and reduction of cytochrome c is related to the reactivity of methionine-80 with mono-iodoacetic acid. The structure-function relationship of cytochrome c is discussed briefly.