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.
A modified form of the Debye-Marcus equation relating electron transfer rate constants to charges on proteins and distances of electron transfer has been applied to the reaction of chemically modified cytochrome f, in which positively charged amino groups are replaced with negatively charged carboxyl groups. The rate of electron transfer from reduced cytochrome f to ferricyanide decreased with increasing ionic strength when the native and singly substituted cytochrome f were used, although a sharp decrease was observed in the former case. When doubly or more than triply substituted cytochrome f was used, the rate of electron transfer was almost constant or increased with increasing ionic strength, respectively. The kinetic-ionic strength effects on this reaction can be well explained by the Debye-Marcus equation in which the charge and radius of the protein are treated as variable parameters. The results show the importance of local positive charges of about 2.0 on native cytochrome f and effective radius of about 11 A of cytochrome f for the electron transfer to ferricyanide. Since the net charge on the native cytochrome f is negative and the calculated radius of the protein is 22.8 A, the above results indicate that positive charges on the electron transfer site control the electrostatic interactions in this reaction. Previously reported data which had been analyzed by using the total net charge and full radius of the protein, were also well explained by the local charge and effective radius of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
The electron transfer reactions of membrane-bound monomeric cytochrome f from Brassica komatosuna (Brassica rapa L. var. perviridis Bailey) with hexacyanoferrate (II)-(III) have been studied as a function of pH, ionic strength and temperature. The second-order rate constant for the oxidation of cytochrome f by Fe(CN)6(3-) at pH 7.0, mu 0.1 M, and 20 degrees C is 1.7 X 10(5) M-1 X S-1, which is similar to the value of oligomeric cytochrome f from parsley. The activation parameters obtained were delta H not equal to = -0.87 kcal/mol and delta S not equal to - -38 cal/mol x deg. Respective rat constant and activation parameters obtained for the reduction of cytochrome f by Fe(CN)6(4-) were k = 1.7 X 10(4) M-1 x S-1, delta H not equal to = +6.7 kcal/mol, and delta S not equal to -16 cal/mol x deg. Both the rate constants for the oxidation and the reduction of cytochrome f markedly decreased with increasing ionic strength. The results indicate that the oxidation and the reduction take place at a positively charged site on the cytochrome f surface, and electrostatic interactions are important for these reactions. The participation of protons and specific amino acid residues in electron transfer reactions of cytochrome f is implied from the pH results. Alkaline isomerization of ferricytochrome f was not observed. The midpoint potential of cytochrome f has a constant value of 360 mV between pH 5.0-8.9, and decreases by about 55 mV per pH unit above 8.9. The results are compared with the data for horse heart cytochrome c and Euglena gracilis cytochrome c-552. These data are discussed in relation to the theories of electrostatic corrected outer-sphere electron transfer of Marcus and multiphonon nonadiabatic electron tunneling of Jortner and Hopfield.
2-p-Toluidino-naphthalene-6-sulfonate is a sensitive fluorescent reporter group which can be used for the detection of the conformation of fructose 1,6-diphosphatase from spinach chloroplasts. When fructose 1,6-diphosphatase was added to a dilute solution of 2-p-toluidino-naphthalene-6-sulfonate at pH 9.0, the fluorescence intensity gradually increased. At this pH, the enzyme activity decreased at the same rate. However, at neutral pH (7.5), this time-dependent fluorescence change was not observed. In the presence of Mg2+, which is an activator of the enzyme, the fluorescence intensity was increased instantly and did not change for 30 min in the pH range 8.0--9.0. From the concentration dependence of the fluorescence intensity, the dissociation constant for Mg2+ was determined, Kdis = 3 mM. The effects of pH and Mg2+ on the conformation and activity of chloroplast fructose 1,6-diphosphatase are discussed.
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