Towards an understanding of redox heterogeneity of the photosystem II cytochrome b559 in the native membrane.

A complex redox titration pattern of cytochrome (Cyt) b559 in preparations of thylakoid membranes and photosystem (PS) II membrane fragments is commonly attributed to the presence of three conformational forms differing by a structure of the heme microenvironment. However, despite decades of research, structural determinants underlying differences between the redox forms of Cyt b559 have not been defined. In this work, we propose a different interpretation of redox heterogeneity in the native population of Cyt b559 assuming redox interaction between the Cyt b559 heme group and a nearby bound quinone (Q). The interacting quinone is supposed to be plastoquinone QC present in the unusual singly protonated form (QCH). The model successfully explains the unique redox properties of Cyt b559 and may provide a simple and effective mechanism of redox regulation of secondary electron transport in PS II. At the present time, the model of heme-quinone redox interaction can be considered as an alternative to the idea of conformational differences between the native redox forms of Cyt b559.

Biphasic reduction of cytochrome b559 by plastoquinol in photosystem II membrane fragments: evidence for two types of cytochrome b559/plastoquinone redox equilibria.

In photosystem II membrane fragments with oxidized cytochrome (Cyt) b559 reduction of Cyt b559 by plastoquinol formed in the membrane pool under illumination and by exogenous decylplastoquinol added in the dark was studied. Reduction of oxidized Cyt b559 by plastoquinols proceeds biphasically comprising a fast component with a rate constant higher than (10s)(-1), named phase I, followed by a slower dark reaction with a rate constant of (2.7min)(-1) at pH6.5, termed phase II. The extents of both components of Cyt b559 reduction increased with increasing concentrations of the quinols, with that, maximally a half of oxidized Cyt b559 can be photoreduced or chemically reduced in phase I at pH6.5. The photosystem II herbicide dinoseb but not 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) competed with the quinol reductant in phase I. The results reveal that the two components of the Cyt b559 redox reaction reflect two redox equilibria attaining in different time domains. One-electron redox equilibrium between oxidized Cyt b559 and the photosystem II-bound plastoquinol is established in phase I of Cyt b559 reduction. Phase II is attributed to equilibration of Cyt b559 redox forms with the quinone pool. The quinone site involved in phase I of Cyt b559 reduction is considered to be the site regulating the redox potential of Cyt b559 which can accommodate quinone, semiquinone and quinol forms. The properties of this site designated here as QD clearly suggest that it is distinct from the site QC found in the photosystem II crystal structure.

Two reaction pathways for transformation of high potential cytochrome b559 of PS II into the intermediate potential form.

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized ("O-type" effects) or when it is reduced ("R-type" effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the "O-type" mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an "R-type" mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as Q(C) which interacts with Cyt b559 and is clearly not the Q(B) site. Binding of compounds L to the Q(C) site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E(m) of HP Cyt b559 with increasing concentration of L (up to 10 K(ox)(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to Q(C) site when HP Cyt b559 is reduced (described by K(red)(L)) induce a conversion of HP Cyt b559 to lower potential redox forms ("R-type" transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the Q(C) site are discussed.

Evidence for a novel quinone-binding site in the photosystem II (PS II) complex that regulates the redox potential of cytochrome b559.

The present study provides a thorough analysis of effects on the redox properties of cytochrome (Cyt) b559 induced by two photosystem II (PS II) herbicides [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,4-dinitro-6-sec-butylphenol (dinoseb)], an acceleration of the deactivation reactions of system Y (ADRY) agent carbonylcyanide-m-chlorophenylhydrazone (CCCP), and the lipophilic PS II electron-donor tetraphenylboron (TPB) in PS II membrane fragments from higher plants. The obtained results revealed that (1) all four compounds selectively affected the midpoint potential (E(m)) of the high potential (HP) form of Cyt b559 without any measurable changes of the E(m) values of the intermediate potential (IP) and low potential (LP) forms; (2) the control values from +390 to +400 mV for HP Cyt b559 gradually decreased with increasing concentrations of DCMU, dinoseb, CCCP, and TPB; (3) in the presence of high TPB concentrations, a saturation of the E(m) decrease was obtained at a level of about +240 mV, whereas no saturation was observed for the other compounds at the highest concentrations used in this study; (4) the effect of the phenolic herbicide dinoseb on the E(m) is independent of the occupancy of the Q(B)-binding site by DCMU; (5) at high concentrations of TPB or dinoseb, an additional slow and irreversible transformation of HP Cyt b559 into IP Cyt b559 or a mixture of the IP and LP Cyt b559 is observed; and (6) the compounds stimulate autoxidation of HP Cyt b559 under aerobic conditions. These findings lead to the conclusion that a binding site Q(C) exists for the studied substances that is close to Cyt b559 and different from the Q(B) site. On the basis of the results of the present study and former experiments on the effect of PQ extraction and reconstitution on HP Cyt b559 [Cox, R. P., and Bendall, D. S. (1974) The functions of plastoquinone and beta-carotene in photosystem II of chloroplasts, Biochim. Biophys. Acta 347, 49-59], it is postulated that the binding of a plastoquinone (PQ) molecule to Q(C) is crucial for establishing the HP form of Cyt b559. On the other hand, the binding of plastoquinol (PQH2) to Q(C) is assumed to cause a marked decrease of E(m), thus, giving rise to a PQH2 oxidase function of Cyt b559. The possible physiological role of the Q(C) site as a regulator of the reactivity of Cyt b559 is discussed.

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants.

"Reduced minus oxidized" difference extinction coefficients Deltavarepsilon in the alpha-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1+/-1.0 mM(-1) cm(-1) and 27.0+/-1.0 mM(-1) cm(-1) were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from -250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E(m) values at pH 6.5 of 244+/-11 mV and -94+/-21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the alpha-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q(B) site and the microenvironment of the heme group of Cyt b559 are discussed.