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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).

Oxygenic photosystem (PS) II complex from spinach photooxidized hydroxyurea (HU) to produce its aminoxy radical, which was identified by its electron spin resonance spectrum. HU was apparently photooxidized by the water-oxidizing enzyme (WOE) since the photooxidation reaction was blocked by carbonyl cyanide m-chlorophenylhydrazone (CCCP). HU radicals photoproduced by the WOE inhibited the electron transfer between the redox-active tyrosine residue (YZ), which is involved in electron transfer from the WOE to the reaction center chlorophyll of PS II, and the secondary quinone electron acceptor. Treatment of PS II complex with Tris resulted in the appearance of a CCCP-insensitive photooxidation site for HU. Photoproduced HU radicals in oxygenic and Tris-treated PS II complex decayed with first-order kinetics, an indication that the radicals reacted primarily with surrounding molecules rather than decayed through spontaneous dismutation or recombination. HU inhibited the diphenylcarbazide-supported photoreduction of 2,6-dichlorophenolindophenol (DCIP) in Tris-treated PS II complex preincubated only under illumination, but this inhibition was suppressed when ascorbate was added to scavenge HU radicals. If examined in darkness, HU radicals could not, however, inhibit subsequent photoreduction of DCIP. Therefore, the photoproduced HU radicals interact with a photogenerated site(s) in the PS II complex. The photoproduction of YZ., a radical of YZ, was suppressed to about 40% in Tris-treated PS II complex by the in situ-photogenerated HU radicals, and the yield of a cation radical of chlorophyll, close to the PS II reaction center, was increased, while the production of a radical of another redox-active tyrosine residue in PS II (YD.) was hardly affected.(ABSTRACT TRUNCATED AT 250 WORDS)