Importance of local positive charges on cytochrome f for electron transfer to plastocyanin and potassium ferricyanide.

To study the relationship between the electron transfer rate and the net or local charge of protein, chemically modified cytochrome f, in which positively charged amino groups are replaced with negatively charged carboxyl groups, has been prepared by using an arylating reagent 4-chloro-3,5-dinitrobenzoic acid. Four distinct species of chemically modified cytochrome f, having 1 to 4 mol of modified amino residues per mol of cytochrome f, were separated by preparative polyacrylamide gel electrophoresis. The rate of electron transfer from the reduced singly substituted cytochrome f to the oxidized spinach plastocyanin was only about 50% of that of the native unmodified cytochrome f. The reaction rate further decreased about 50% upon the modification of each amino residue. The biphasic oxidation of cytochrome f by plastocyanin was observed when more than 2 mol of amino residues were modified. The rate of the second phase also decreased with an increasing number of modified amino residues. On the other hand, the oxidation of chemically modified cytochrome f by potassium ferricyanide was clearly monotonic. The rate decreased about 30% upon the modification of each amino residue. The midpoint potentials of chemically modified cytochrome f were almost the same as that of the native protein. These results clearly indicate the importance of local positive charges on cytochrome f, since the overall net charge of cytochrome f is negative at neutral pH. The theory of electrostatic corrected outer-sphere electron transfer of Marcus explained the effect of charge on cytochrome f for the reaction with the small molecule of ferricyanide well, but not the reaction with the protein of plastocyanin.