Intermediate Potential Research Articles

Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b559 in Tris-treated Photosystem II membranes

The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b 559 (cyt b 559) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HP Fe2+) form of cyt b 559, whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HP Fe3+) form of cyt b 559. Light-induced conversion of cyt b 559 from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b 559 after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b 559 proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b 559 depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b 559 is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.

Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b 559 (cyt b 559) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP red) forms of cyt b 559 exhibit superoxide scavenging activity and interconversion between IP and HP red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O 2 − ) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O 2 − scavengers prevented the light-induced conversion of IP ↔ HP red cyt b 559 confirmed that IP and HP red cyt b 559 are reduced and oxidized by O 2 − , respectively. Redox changes in cyt b 559 by an exogenous source of O 2 − reconfirmed the superoxide oxidase and reductase activity of cyt b 559. Furthermore, the light-induced conversion of IP to HP red form of cyt b 559 was completely inhibited at pH > 8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His 22 residue generates a favorable condition for the oxidation and reduction of O 2 − , respectively.

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.

Restoration of the high-potential form of cytochrome b559 of photosystem II occurs via a two-step mechanism under illumination in the presence of manganese ions

Spinach photosystem II membranes that had been depleted of the Mn cluster contained four forms of cytochrome (Cyt) b559, namely, high-potential (HP), HP′, intermediate-potential (IP) and low-potential (LP) forms that exhibited the redox potentials of +400, +310, +170 and +35 mV, respectively, in potentiometric titration. When the membranes were illuminated with flashing light in the presence of 0.1 mM Mn 2+, the IP form was converted to the HP′ form by two flashes and then the HP′ form was converted to the HP form by an additional flash. The quantum efficiency of the first conversion appeared to be quite high since the conversion was almost complete after two flashes. By contrast, the second conversion proceeded with low quantum efficiency and 40 flashes were required for completion. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suggested that the first conversion did not require electron transfer from Q A to Q B while the second conversion had an absolute requirement for it. It was also suggested that the first conversion involved the reduction of the heme of Cyt b559, probably by Q A −, and we propose that direct reduction by Q A − induces a shift in the redox potential of the heme. The second conversion was also accompanied by the reduction of heme but it appeared that this conversion did not necessarily involve the reduction. The effects of DCMU on the reduction of heme suggested that the heme became reducible by Q B − after the first conversion had been completed. This observation implies that the efficiency of electron transfer from Q A to Q B increased upon the conversion of the IP form to the HP′ form, and we propose that restoration of the high-potential forms of Cyt b559 itself acts to make the acceptor side of photosystem II functional.