Binding of Novel Inhibitors of Electron Transfer in Photosystem 2, Derivatives of Perfluoroisopropyldinitrobenzene, with Polypeptide D2 of the Reaction Center

S. K. Zharmukhamedov¹, M. S. Kristin¹, Li Shuqin², S. I. Allakhverdiev¹, and V. V. Klimov^1*

¹Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia; E-mail: klimov@issp.serpukhov.su

²Institute of Botany, Chinese Academy of Sciences, 100093 Beijing, China; E-mail: lishuqin1965@yahoo.com.cn

^* To whom correspondence should be addressed.

Received July 31, 2002

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A binding site for novel inhibitors of K15 type (derivatives of perfluoroisopropyldinitrobenzene) with the components of reaction center (RC) of photosystem 2 (PS-2) of higher plants has been investigated. It has been shown that multiple washing the PS-2 submembrane chloroplast fragments (BBY-particles) treated with the K15 inhibitor, including multiple dilution in buffer in the presence of high concentrations of mono- and divalent ions, prolonged (up to 2-5 h) incubation, centrifugation, and subsequent resuspension in buffer deprived of the inhibitor, does not lead to restoration of functional activity of the PS-2. After addition of dithionite, inducing reduction and consequent decomposition of the inhibitor, and subsequent removal of dithionite by washing, the functional activity of PS-2 was completely restored. Incubation in the presence of sodium dodecyl sulfate (SDS), leading to solubilization of the sample to the level of protein components, induced the appearance of a fraction of free K15 retaining the initial inhibitory efficiency. To create a covalent binding of the inhibitor with protein, retained under the conditions of denaturing SDS polyacrylamide gel electrophoresis, the azido-containing analog of K15 (K15-N₃) was used. The need for radioactive label for identification of K15 was avoided by the revealed ability of K15-type inhibitors to emit fluorescence, which retained its features under the experimental conditions. With the technique of photoaffinity binding and denaturing SDS-PAGE in the presence of 6 M urea of submembrane chloroplast fragments enriched in PS-2 the D2-polypeptide, an integral component of the reaction center of PS-2, has been shown to be a binding site for K15-type inhibitors. This conclusion is in agreement with a suggestion (put forward in our earlier publications) that K15-type inhibitors are bound to PS-2 reaction center, replacing Q_A in its binding site. Hence, an agent specifically binding to polypeptide D2 has been found for the first time. The data are compared with information about inhibitory action and binding sites of the known inhibitors of electron transfer in PS-2.

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KEY WORDS: photosystem 2, electron phototransfer, electron transport inhibitors, binding site

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