The Research Institute for Food Science, Kyoto University, 611, Uji, Kyoto, Japan.
Photosynth Res. 1993 Jan;38(3):433-40. doi: 10.1007/BF00046771.
A new binding site for anions which inhibit the water oxidizing complex (WOC) of Photosystem II in spinach has been identified. Anions which bind to this site inhibit the flash-induced S2/S0 catalase reaction (2H2O2→2H2O+O2) of the WOC by displacing hydrogen peroxide. Using a mass spectrometer and gas permeable membrane to detect the (32)O2 product, the yield and lifetime of the active state of the flash-induced catalase (to be referred to simply as 'flash-catalase') reaction were measured after forming the S2 or S0-states by a short flash. The increase in flash-catalase activity with H2O2 concentration exhibits a Km=10-20 mM, and originates from an increase in the lifetime by 20-fold of the active state. The increased lifetime in the presence of peroxide is ascribed to formation of the long-lived S0-state at the expense of the unstable S2-state. The anion inhibition site differs from the chloride site involved in stimulating the photolytic water oxidation reaction (2H2O→O2+4e(-)+4H(+)). Whereas water oxidation requires Cl(-) and is inhibited with increasing effectiveness by F(-)≪CN(-)≪N3 (-), the flash-catalase reaction is weakly inhibited by Cl(-), and with increasing effectiveness by F(-)≪CN(-), N3 (-). Unlike water oxidation, chloride is unable to suppress or reverse inhibition of the flash-catalase reaction caused by these anions. The inhibitor effectiveness correlates with the pKa of the conjugate acid, suggesting that the protonated species may be the active inhibitor. The reduced activity arises from a shortening of the lifetime of the flash-induced catalase active state by 3-10 fold owing to stronger anion binding in the flash-induced states, S2 and S0, than in the dark S-states, S1 and S-1. To account for the paradoxical result that higher anion concentrations are required to inhibit at lower H2O2 concentrations, where S2 forms initially after the flash, than at higher H2O2 concentrations, where S0 forms initially after the flash, stronger anion binding to the S0-state than to the S2-state is proposed. A kinetic model is given which accounts for these equilibria with anions and H2O2. The rate constant for the formation/release of O2 by reduction of S2 in the WOC is <0.4 s(-1).
已鉴定出一种新的阴离子结合位点，该位点可抑制菠菜光合系统 II 的水氧化复合酶（WOC）。与该位点结合的阴离子通过取代过氧化氢来抑制 WOC 的闪光诱导 S2/S0 过氧化氢酶反应（2H2O2→2H2O+O2）。使用质谱仪和透气膜检测（32）O2 产物，通过短闪光形成 S2 或 S0 态后，测量闪光诱导过氧化氢酶（简称为“闪光-过氧化氢酶”）反应的活性态的产率和寿命。随着 H2O2 浓度的增加，闪光-过氧化氢酶活性的增加呈现 Km=10-20mM，并且源自活性态寿命增加 20 倍。过氧化物存在下寿命的增加归因于稳定的 S2 态形成，而不稳定的 S0 态则形成。阴离子抑制位点不同于参与刺激光解水氧化反应（2H2O→O2+4e(-)+4H(+））的氯位点。虽然水氧化需要 Cl(-），并且随着 F(-）≪CN(-）≪N3 (-）的有效性增加而受到抑制，但氯(-）对闪光-过氧化氢酶反应的抑制作用较弱，而 F(-）≪CN(-），N3 (-）的抑制作用则增强。与水氧化不同，氯离子无法抑制或逆转这些阴离子对闪光-过氧化氢酶反应的抑制作用。抑制剂的有效性与共轭酸的 pKa 相关，表明质子化物质可能是活性抑制剂。由于在闪光诱导的 S2 和 S0 态中阴离子结合比在暗态 S1 和 S-1 中更强，因此活性状态的闪光诱导过氧化氢酶的寿命缩短了 3-10 倍，导致活性降低。为了解释在较低 H2O2 浓度下需要更高的阴离子浓度才能抑制初始形成 S2 的初始形成的闪光后，而在初始形成 S0 的较高 H2O2 浓度下需要更高的阴离子浓度才能抑制的矛盾结果，提出了阴离子与 S0 态的结合比与 S2 态更强的观点。提出了一个动力学模型，该模型用阴离子和 H2O2 来解释这些平衡。WOC 中 S2 还原形成/释放 O2 的速率常数<0.4 s(-1）。
