The protein-protein electron transfer reactions between cytochrome f and plastocyanin, both purified from Brassica komatsuna (Brassica rapa L. var. pervirdis Bailey), have been studied as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of ferrocytochrome f by plastocyanin was found to be k = 4.5 X 10(7) M-1 x S-1 at pH 7.0 mu 0.2 M, and 20 degrees C, with activation parameters delta H not equal to = 8.4 kcal/mol and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions are attributable not to a low activation enthalpy but to a positive activation entropy term. The rate constants both for the oxidation and the reduction of cytochrome f by plastocyanin drastically decreased with increasing ionic strength, indicating the importance of electrostatic interactions. Divalent cations are more effective than monovalent cations in reducing the rates of these reactions. The rate constants for the oxidation of cytochrome f by plastocyanin are constant between pH 6.0 and 9.0 but decrease markedly above pH 9.0 and below pH 6.0. In the case of the reduction of cytochrome f by plastocyanin, an optimum pH around 7.0 was obtained and a biphasic feature was observed at alkaline pH. The results are discussed in relation to photosynthetic electron transport systems.

The rates of the reduction of Nitroblue Tetrazolium by O2− during the autoxidation of pyrogallol, epinephrine, and catechol were increased 110, 55, and 37-fold respectively in the presence of hexadecyltrimethylammonium bromide. A nonionic micelle of polyoxyethylene dodecyl ether also accelerated these reactions. The microenvironment effects on the electron-transfer from O2− to Nitroblue Tetrazolium were discussed.

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.

In the absence of EDTA, the rate of autoxidation of dithiothreitol (2•~10-5M) increased with an increase of the concentration of Cu2+, and reached to the maximum at 5•~10-8 M Cu2+. And then the rate declined and the autoxidation of dithiothreitol ceased at 1.6•~10-8 M Cu2+ In the presence of EDTA, both the rate and the optimum concentration of Cul+ increased. A model which involves Cu2+-EDTA or Cu2+-(dithiothreitol)2 and 02 complexes for the catalysis of the autoxidation of dithiothreitol was proposed.

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