Biochemistry zyxwvut 1995,34, zyxwvu 5839-5858 5839 Amino Acid Residues That Influence the Binding of Manganese or Calcium to Photosystem 11. 1. The Lumenal Interhelical Domains of the D1 Polypeptide? Hsiu-An Chu, Anh P. Nguyen, and Richard J. Debus* Department of Biochemistry, University zyxwvut of California at Riverside, Riverside, California zyxw 92521-0129 Received November 1, 1994; Revised Manuscript Received February 22, 1995@ ABSTRACT: To identify amino acid residues that ligate the manganese and calcium ions of photosystem I1 or that are otherwise crucial to water oxidation, site-directed mutations were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803 at all conserved carboxylate, histidine, and tyrosine residues in the lumenal interhelical domains of the D1 polypeptide. Mutants with impaired photoautotrophic growth or oxygen evolution were characterized in zyxwvutsr vivo by measuring changes in the yield of variable chlorophyll zy a fluorescence after a saturating flash or brief illumination given in the presence of an electron-transfer inhibitor or following each in a series of saturating flashes given in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., zyxwvutsrq & Debus, R. J. (1994) Biochemistry 33, 6137-61491. Mutants were also characterized after propagation in media having other cations substituted for calcium. We conclude that Asp-59 and Asp-61 may ligate calcium, that Asp-59, Asp-61, Glu-65, and His-92 influence the properties of the manganese cluster without significantly affecting its stability or ability to assemble, that Glu- 189 plays an important structural role in maintaining the catalytic efficiency of the Mn cluster and partly influences the cluster's stability or ability to assemble, that His-92 and Glu-189 influence the binding of calcium, and that His-190 strongly influences the redox properties of the secondary electron donor, zyx YzoX, and either ligates manganese or serves as a crucial base or hydrogen bond donor. In addition, we conclude that Asp-170 may ligate manganese, but that its replacement that partly compensate for the loss of the carboxylate The oxygen-evolving complex of photosystem I1 (PSII)' contains a cluster of four manganese ions. This cluster catalyzes the oxidation of two molecules of water, releasing one molecule of 0 2 as a byproduct [for reviews, see Rutherford et al. (1992), Debus (1992), and Renger (1993)l. Chloride and one to two calcium ions are required for catalysis and are located near the Mn cluster. The major subunits of the PSII reaction center include an extrinsic polypeptide of 33 kDa and the membrane-spanning proteins CP47, CP43, D1, and D2 [for reviews, see Andersson and Styring (1991), Ikeuchi (1992), and Vermaas et al. (1993)l. Smaller subunits include the a and p polypeptides of cytochrome b-559 and the product of the psb1 gene. The D1 and D2 polypeptides are each believed to contain five membrane-spanning a-helices and to form a heterodimer that contains the primary electron-transfer components of PSII. This work was funded by the National Institutes of Health (GM 43496). * Author to whom correspondence should be addressed. @ Abstract published in Advance ACS Absrrucfs, April 15, 1995. I Abbreviations: A730, optical density at 730 nm; bp, base pair; Chl, chlorophyll a; DCBQ, zyxwvutsrq 2,6-dichloro-p-benzoquinone; DCMU, 3-(3,4- dichloropheny1)-1,1 -dimethylurea; Em', erythromycin resistant; Feq, steady-state fluorescence yield produced by weak monitoring flashes in the presence of DCMU; kb, kilobase; MES, 2-morpholinoethane- sulfonic acid; P680, primary chlorophyll electron donor; PCR, polym- erase chain reaction; PSII, photosystem 11: QA, primary plastoquinone electron acceptor; QB. secondary plastoquinone electron acceptor; S,, oxidation state of the oxygen-evolving complex with n oxidizing equivalents stored; TES, N-[tris(hydroxymethyl)methyl]-2-aminoeth- anesulfonic acid; wild-type*, control Synechocysris strain constructed in the same manner as site-directed mutants, but with no mutation; Yz, rapid electron donor to P680+ (Tyr-161 of the D1 polypeptide): YD, slow electron donor to P680+ (Tyr-160 of the D2 polypeptide). 0006-2960/95/0434-5839$09.00/0 with Val, Leu, or Ile causes structural perturbations moiety. Several lines of evidence suggest that the D1 polypeptide contributes many of the amino acid residues that coordinate the Mn and Ca2+ ions in PSII [for a review, see Debus (1992)l. On the basis of considerations of manganese coordination chemistry, the Mn ions are believed to be coordinated primarily by carboxylate residues, although serine, threonine, tyrosine, and histidine residues may participate (Pecoraro, 1988; Brudvig & Crabtree, 1989; Wieghardt, 1989). Coordination by carboxylates has been supported by chemical modification studies (Tamura et al., 1989a, 1992; Preston & Seibert, 1990, 1991a,b; Blubaugh & Cheniae, 1992) and Fourier transform infrared studies (Noguchi et al., 1992, 1993). Coordination by one or two histidine imidazole nitrogens recently has been demonstrated by ESEEM (electron spin echo envelope modulation) measurements (Tang et al., 1994a), extending earlier ESEEM (DeRose et al., 1991; Zimmermann et al., 1993) and ENDOR (electron nuclear double resonance) studies (Tang et al., 1993) that strongly implicated nitrogen ligation. Coordina- tion by histidine had been proposed earlier on the basis of chemical modification studies (Tamura et al., 1989a; Seibert et al., 1989; Preston & Seibert, 1989, 1990, 1991a,b; Ono & Inoue, 1991) and other considerations (Kambara & Govindjee, 1985; Padhye et al., 1986). Specific amino acid residues have been proposed as ligands to Mn or Ca2+ on the basis of models of the structure of the lumenal regions of the D1D2 heterodimer (Svensson et al., 1990, 1991, 1992; Ruffle & Nugent, 1992; Ruffle et al., 1992) and chemical modification and proteolysis studies (Seibert et al., 1989; Preston & Seibert, 1990, 1991b). As an alternative approach toward the identification of amino acid residues that ligate the Mn and Ca2+ ions in PSII, 1995 American Chemical Society