Biochemistry 1992,31, 8449-8454 8449 Comparative Properties of Hydroquinone and Hydroxylamine Reduction of the Ca2+-Stabilized 02-Evolving Complex of Photosystem 11: Reductant-Dependent Mn2+ Formation and Activity Inhibition? Rui Mei and Charles F. Yocum’ Departments of Biology and Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1048 Received March 4, 1992; Revised Manuscript Received June 26, 1992 ABSTRACT: Calcium binding to photosystem I1 slows NH2OH inhibition of 02 evolution; Mn2+is retained by the 02-evolving complex [Mei, R., & Yocum, C. F. (1991) Biochemistry 30, 7836-78421. This Ca2+- induced stability has been further characterized using the large reductant hydroquinone. Salt-washed photosystem I1 membranes reduced by hydroquinone in the presence of Ca2+ retain 80% of steady-state 02 evolution activity and contain about 2 Mn2+/reaction center that can be detected at room temperature by electron paramagnetic resonance. This Mn2+ produces a weak enhancement of H2O proton spin-lattice relaxation rates, cannot be easily extracted by a chelator, and is reincorporated into the 02-evolving complex upon illumination. A comparison of the properties of Ca2+-supplemented photosystem I1 samples reduced by hydroquinone or NH2OH alone or in sequence reveals the presence of a subpopulation of manganese atoms at the active site of H2O oxidation that is not accessible to facile hydroquinone reduction. At least one of these manganese atoms can be readily reduced by NH2OH following a noninhibitory hydroquinone reduction step. Under these conditions, about 3 Mn2+/reaction center are lost and O2 evolution activity is irreversibly inhibited. We interpret the existence of distinct sites of reductant action on manganese as further evidence that the Ca2+-binding site in photosystem I1 participates in regulation of the organization of manganese-binding ligands and the overall structure of the 02-evolving complex. Water oxidation by PSIS is postulated to occur by successive formation of five oxidation states, Si (i = 04); Sq decays spontaneously to produce SO and 02 (Kok et al., 1970). Four atoms of Mn form the redox-active site of the 02-evolving complex; Ca2+and C1- are also required for activity (Amesz, 1983; Babcock, 1987; Brudvig et al., 1989; Ghanotakis & Yocum, 1990). A requirement for added Ca2+to restore 02 evolution activity in PSII preparations is observed when extrinsic 23- and 17-kDa polypeptides are removed from PSII (Ghanotakis et al., 1984a; Miyao & Murata, 1984; Naka- tani, 1984). It has been proposed that these polypeptides and an extrinsic 33-kDa species provide part of a structure that concentrates inorganic ion cofactors at the site of H2O oxidation (Ghanotakis et al., 1984b). The observation that Ca2+ extraction from PSII disrupts electron transfer from Mn to Yz+ suggests that the metal is required for S-state cycling (Dekker et al., 1984; Kalosaka et al., 1990). Analyses of delayed fluorescence yields from PSII indicated that Ca2+ extraction impedes the S3 - Sq transition (Boussac et al., 1985),and some EPR measurementssuggested that the SI - S2 transition is affected by Ca2+removal (de Paula et al., 1986; Ghanotakis et al., 1987; Kalosaka et al., 1990). However, if Ca2+ extraction by EGTA, EDTA, or citrate is carried out in the light, a modified S2 EPR multiline f Research supported by a grant (88-37130-3546) to C.F.Y. from the Competitive Research Grants Office, Photosynthesis Program, of the United States Department of Agriculture. 1 Abbreviations: Chl, chlorophyll; DCBQ, 2,6-dichloro-p-benzoqui- none; EDTA, ethylenediaminetetraacetate; EGTA, ethylene glycol bis- (8-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; EPR, electron para- magnetic resonance; H2Q, hydroquinone; MES, 2-(N-morpholino)- ethanesulfonic acid; Mn, manganeseligated to photosystem I1 in oxidation states higher than +2; NMR, nuclear magnetic resonance; PS, photo- system; TMPD, N,N,”,N’-tetramethyl-p-phenylenediamine; XANES, X-ray absorption near-edge structure; Yz+, redox-active tyrosine 16 1 on the D1 polypeptide of photosystem 11. 0006-2960/92/043 1-8449$03.00/0 signal is observed (Boussac et al., 1989), and thermolumi- nescence measurements on samples from which Ca2+ has been extracted in darkness at pH 3 also show that a modified form of S2 is formed (On0 & Inoue, 1989). The disparities surroundingCa2+ involvement in S2 formation may be due to an increased low-temperature limit for S2 formation, induced byCa2+ extraction (On0 & Inoue, 1990;Boussacet al., 1990). The EPR-detectable S2 multiline signal is not observed after illumination of C1--depleted PSII; subsequent dark addition of C1- produces the signal (On0 et al., 1986). Substitution of F- for C1- generates the g = 4.1 precursor to the multiline signal (Casey & Sauer, 1984),and NH3 can produce a similar result if the C1- concentrationis suppressed (Beck & Brudvig, 1986). Chloride sensitivity of ligand binding on or near Mn in PSII defines two types of sites. Small ligands (NH3, NH2- OH) bind at a C1--insensitive site (Sandusky & Yocum, 1984; Beck & Brudvig, 1986; Mei & Yocum, 1990,1991) whereas a second, C1--sensitive site binds NH3 and larger primary amines, as well as N-methyl derivatives of NH20H and large species such as PD or TMPD (Sandusky & Yocum, 1984; Tamura et al., 1986; Beck & Brudvig, 1988; Mei & Yocum, 1990; Rickert et al., 1991). Exposure of PSII to the small reductant NH2OH can produce two effects. Concentrations greater than 0.1-0.2 mM reduce the Mn complex with a loss of activity and of three to four EPR-detectable Mn2+per reaction center (Cheniae & Martin, 1971; Yocum et al., 1981). An enhancement of H2O proton spin-lattice relaxation rates, attributed to weak binding of Mn2+ to nonfunctional sites outside the 02-evolvingcomplex, is also observed (Sharp & Yocum, 1981). Lower NH2OH concentrations (<<lo0 pM) generate a S-1 state from which 02 release is delayed by two flashes (Bouges, 1971; Kok & Velthuys, 1977). Steady-state illumination of thylakoids incubated with NH2OH consumes the reductant and prevents inhibitory Mn2+ release (Sharp & Yocum, 1981); flash 0 1992 American Chemical Society