Two-electron reactions S2QB -->S0QB and S3QB -->S1QB are involved in deactivation of higher S states of the oxygen-evolving complex of Photosystem II

Abstract

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S(0)-S(4)), and dark incubation leads to 25% S(0) and 75% S(1). This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q(B)(-). We measured flash-induced oxygen evolution to understand how S(3) and S(2) are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q(B)(-). Thylakoid samples preconditioned to make the concentration of the S(1) state 100% and to oxidize tyrosine Y(D) were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S(2) and S(3) are converted to lower S states via recombination from S(2)Q(B)(-) and S(3)Q(B)(-) and by a slow change of the state of oxygen-evolving complex from S(3) and S(2) to S(1) and S(0) in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S(2)Q(B) -->S(0)Q(B), and S(3)Q(B) -->S(1)Q(B). The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S(3)Q(B) -->S(2)Q(B) -->S(1)Q(B) in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H(2)O(2) acts as an electron donor for the reaction S(2)Q(B) -->S(0)Q(B) but added H(2)O(2) did not enhance this reaction.

Figure 1

Scheme of S state transitions induced by one or two preflashes (A) and transitions occurring during consequent dark deactivation in PS II centers according to the classical one-electron-per-step mechanism (B) and according to the proposed two-electron mechanism (C). Before the preflashes were fired, all PS II centers were assumed to be in the S₁Y_D^ox state with 20% Q_B⁻ and 80% Q_B. In B and C, one-electron and two-electron reactions occurring during deactivation of states S₂Q_B and S₃Q_B are shown with solid and dashed arrows, respectively, and charge recombination of pairs S₂Q_B⁻ and S₃Q_B⁻ is indicated by dotted arrows.

Figure 2

Flash-induced O₂ evolution patterns measured in S₁Y_D^ox thylakoid preparations (solid symbols) and the best fit (open symbols) calculated by using the one-miss model with all misses occurring in the S₃ → S₀ transition. S₁Y_D^ox thylakoids were treated with two preflashes, and the O₂ evolution pattern was then measured after 2 (curve 1) or 10 (curve 2) min of dark incubation. Amplitudes of O₂ evolution are normalized to the maximal value in the train. (Inset) Flash-induced O₂ evolution measured under the same conditions as in curve 2 in the presence or absence of 1 mM FeCy, as indicated. The data show typical flash-induced O₂ evolution patterns.

Figure 3

Deactivation kinetics of S-states after one (A) or two (B) preflashes in S₁Y_D^ox thylakoid membranes. The data show the percentage of the sum of all S states: S₀ + S₁ + S₂ + S₃ = 100%. Each data point represents the average of three repeats, and error bars show standard deviation. Fitting was done using the one-electron-per-step model for the deactivation of the higher S-states (dotted lines, the model is shown in Fig. 1B) and using the two-electrons-per-step model (solid lines, the model is shown in Fig. 1C). (Inset) Decay of delayed light emission after one flash in S₁Y_D^ox thylakoids.

Figure 4

Deactivation kinetics of S states after two preflashes in S₁Y_D^ox thylakoid membranes in the presence of 250 μM DCBQ. The data show the percentage of the sum of all S-states: S₀ + S₁ + S₂ + S₃ = 100%. Fitting was done using the one-electron-per-step model for the deactivation of the higher S states (dotted lines) and using the two-electrons-per-step model (solid lines) (see models in Fig. 1 B and C). Both models were extended by including the reaction S₁Q_B → S₀Q_B. The kinetic pattern is from one representative experiment.

Figure 5

Production of hydrogen peroxide in thylakoids during 10 min dark incubation under different conditions. (A) Production of H₂O₂ in dark-adapted S₁Y_D^ox thylakoids incubated without additives (control), with catalase (30 U in 100 μL), under argon flow, and after treatment with 5 mM sodium azide. (B) Production of H₂O₂ in S₁Y_D^ox thylakoids during 10 min dark incubation of nonilluminated samples and after illumination of the sample with one or two flashes as indicated. The measurements were carried out in the presence (dark gray) or absence (light gray) of 0.4 μM CCCP, added 10 s after the preflash(es) were fired. H₂O₂ was measured with the Amplex Ultrared method. Each bar represents an average of three independent experiments and the error bars show standard deviation.