The reaction of Rhodobacter sphaeroides cytochrome c2 with the Rb. sphaeroides cytochrome bcl complex was studied by using singly labeled cytochrome c2 derivatives. Cytochrome c2 was treated with chlorodinitrobenzoic acid to modify lysine amino groups to negatively charged carboxydinitrophenyllysines and separated into eight different fractions by ion-exchange chromatography on a Whatman S E 53 (sulf-oxyethy1)cellulose column. Peptide mapping studies indicated that six of these fractions were

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... dified at single lysine amino groups. Each of the derivatives had the same V, , , value as native cytochrome c2 in the steady-state reaction with the Rb. sphaeroides cytochrome bc, complex. However, the K, values of the cytochrome c2 derivatives modified a t lysines 10, 55, 95, 97, 99, and 106 were found to be larger than that of native cytochrome c2 by factors of 6, 2, 3, 32, 13, and 8, respectively. These results indicate that lysines located in the sequence 97-106 on the left side of the heme crevice have the greatest involvement in binding the cytochrome bcl complex. The involvement of lysine 97 is especially significant because it