Cytochrome c2 and the detergent-solubilized cytochrome bc1 complex, both from Rhodospirillum rubrum, form a tight complex at a low ionic strength that can be isolated by gel permeation chromatography. The dissociation constant of the complex is estimated to be 10–6 M or less. The binding site for the cytochrome bc1 complex on cytochrome c2 was analyzed by differential acetylation of lysine residues in free and cytochrome bc1 complex bound cytochrome c2. In bound cytochrome c2, three lysine residues at sequence positions 12, 13, and 97 were less reactive toward acetic anhydride. Lys13, which is located above the exposed heme edge, was the least reactive, i.e., the most shielded by the cytochrome bc1 complex. Correlating this information with the crystal structure of cytochrome c2 indicates that the binding site for the cytochrome bc1 complex on cytochrome c2 involves a surface area above, and probably including, the exposed heme edge. This mode of binding is similar to that observed for horse cytochrome c interacting with the mitochondrial cytochrome bc1 complex. A simplified version of the method of differential chemical modification is presented.
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