photosystem II particles from Synechococcus sp., 2-AcOMeNQ primarily labels two polypeptides with apparent molecular masses of 38 and 19 kDa. Labeling of ...
Proc. Nati. Acad. Sci. USA Vol. 84, pp. 1774-1778, April 1987 Biochemistry
Labeling quinone-binding sites in photosynthetic reaction centers: A 38-kilodalton protein associated with the acceptor side of photosystem II (active-site mapping/cofactor/sequence homology)
STEPHEN T. WORLAND*, AKIHIKO YAMAGISHIt, STEPHEN ISAACS, KENNETH SAUER, AND JOHN E. HEARST Department of Chemistry and Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720
Communicated by George C. Pimentel, November 12, 1986
The sequence homologies between bacterial and PSII reaction centers led us to undertake experiments aimed at labeling quinone-binding sites. Previously, experimenters have labeled quinone-binding sites in bacterial RCs (18), ubiquinol-cytochrome c reductase from beefheart mitochondria (19), and the cytochrome b6-f complex from spinach chloroplasts (20). In related work, herbicide-binding sites have been labeled in bacterial RCs (21, 22) and PSII RCs from spinach (11, 23, 24). We chose a class of quinone derivatives that had been suggested to be mechanism-based inhibitors of quinone-binding enzymes-i.e., quinone derivatives possessing a latent alkylating potential that is activated by reduction of the quinone (25). In our hands, the reagents tested proved to be active alkylating agents prior to any reduction and showed no further activation by reduction. We were still able to use one such derivative to specifically label quinone-binding sites. This was possible because the compound 2-acetoxymethyl-1,4-naphthoquinone (2-AcOMeNQ) binds to RCs and PSII preparations with a high affinity and because the kinetics of binding is considerably faster than the rate of alkylation. We labeled quinone-binding sites in RCs from Rhodopseudomonas capsulata and in PSII RCs from the thermophilic cyanobacterium Synechococcus sp. We interpret the labeling pattern of R. capsulata RCs in terms of the recently published crystal structure of a closely related RC from Rhodopseudomonas viridis (9) and extend the analysis to suggest modes of quinone binding in PSII RCs.
2-Acetoxymethyl-1,4-naphthoquinone (2ABSTRACT AcOMeNQ) binds with rapid kinetics and high affinity to the primary quinone QA site of reaction centers from Rhodopseudomonas capsulata. Binding of 2-AcOMeNQ fully restores electron-transfer activity with kinetics that is similar, but not identical, to that seen with ubiquinone-50. When bound at the QA site, 2-AcOMeNQ preferentially labels the L subunit. This preference suggests that 2-AcOMeNQ labels primarily the region of a quinone-binding site that is close to the first isoprenoid unit of the side chain, which is expected from the location and structure of the reaction region of the molecule. In photosystem II particles from Synechococcus sp., 2-AcOMeNQ primarily labels two polypeptides with apparent molecular masses of 38 and 19 kDa. Labeling of only the 38-kDa polypeptide is sufficiently sensitive to 3-(3,4-dichlorophenyl)1,1-dimethylurea (DCMU) to conclude that it is involved in binding quinones on the acceptor side of photosystem II. Although we have not yet identified the 38-kDa protein, its properties suggest that it is the D2 protein. From the DCMUsensitive labeling and from homologies to functionally important regions of the bacterial reaction-center subunits, we propose that the 38-kDa protein is intimately involved in binding the cofactors that mediate primary photochemistry.
Photosynthetic reaction centers (RCs) are integral membrane proteins that bind several cofactors capable of participating in electron-transfer reactions. RCs convert electromagnetic radiation into chemical potential via a rapid electron transfer from an excited state of bacteriochlorophyll BChl to an adjacent acceptor. The initial charge-separated state is stabilized by subsequent transfer of the electron to other acceptors within the RC. RCs from purple nonsulfur bacteria share a number of similarities with photosystem II (PSII) RCs from higher organisms, including the presence of related bound cofactors (1). The L and M subunits of bacterial RCs have limited regions of sequence homology to the D1 and D2 proteins associated with PSII (2-8). Several of the bacterial RC residues that are homologous to D1 and D2 are in intimate contact with the cofactors that mediate primary photochemistry (9, 10). The roles of the D1 and D2 proteins in electron transport through PSII are of considerable current interest. D1 is the site of action of a number of herbicides (11) and is believed to bind the secondary quinone QB (12). The role of D2 is less clear right now. The fact that D1 and D2 show homologies to functionally important regions of M and L has led to proposals that in PSII D1 and D2 bind the cofactors responsible for mediating primary photochemistry (9, 10, 13, 14). Alternative proposals assign this role to the 47- and/or 43-kDa proteins (15-17).
METHODS RCs from R. capsulata strain KZR8A1 (26) were purified as described (27, 28). RC concentrations were determined by using the extinction coefficient for the RC from Rhodopseudomonas sphaeroides strain R-26 (29). Electron-transfer reactions were monitored by observing absorbance changes (AA) at 595 nm ("bleaching signal") induced by actinic illumination of the sample with near IR light (750-900 nm). To avoid irreversible bleaching, the saturated bleaching signal was obtained by extrapolating from nonsaturating conditions. The extrapolation can be done from a double-reciprocal plot of 1/AA vs. 1/actinic light intensity (1/I) (30), but a statistically more significant extrapolation is obtained from a single-reciprocal plot of AA vs. AA/I. In this type of plot, the saturated bleaching signal is the y intercept, and the negative slope of the line is directly related to the value of I required to half-saturate the bleaching. The half-saturating intensity is Abbreviations: Chl, chlorophyll; BChl, bacteriochlorophyll; PSII, photosystem II; 2-AcOMeNQ, 2-acetoxymethyl-1,4-naphthoquinone; I, actinic light intensity; UQ-50, ubiquinone-50; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; RC, reaction center; E, einstein (1 mol of photons). *Present address: Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138. tPresent address: Tokyo Institute of Technology, Tokyo, Japan.
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Biochemistry: Worland et al. related to the rates of formation and decay of the chargeseparated state (30) and thus provides a sensitive measure of the rates of electron-transfer reactions within the RC. Occupancy at the QB site was determined by the presence of a slow (rate constant < 1 s-1) kinetic phase in the decay of the bleaching signal (31). Occupancy at the QA site was determined by comparing the magnitude of the bleaching signal to the bleaching in the presence of saturating amounts of ubiquinone-50 (UQ-50). Quinones were extracted from RCs by the method of Okamura et al. (32), except that RCs were 2.5 ,uM, o-phenanthroline was 5 mM, and lauryldimethylamine oxide was 0.5%. After extraction, occupancy at the QA site varied between 20% and 40% for different preparations. RCs were reconstituted with UQ-50 or 2-AcOMeNQ by addition of the quinone in ethanol or acetone [final ethanol concentration,
