Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7099-7103, October 1987 Biophysics Tyrosine radicals are involved in the photosynthetic oxygen- evolving system BRIDGETTE A. BARRY AND GERALD T. BABCOCK Department of Chemistry, Michigan State University, East Lansing, MI 48824-1322 Communicated by N. E. Tolbert, June 15, 1987 (received for review May 14, 1987) ABSTRACT In addition to the reaction-center chloro- same chemical structure. This assumption is reinforced by phyll, at least two other organic cofactors are involved in the the distinctive nature of the EPR signal, particularly its photosynthetic oxygen-evolution process. One of these cofac. partially resolved hyperfine coupling and its g value of tors, called "Z," transfers electrons from the site of water 2.0046. The hyperfine structure arises from an interaction oxidation to the reaction center of photosystem II. The other between the unpaired electron and protons, since the Dt species, "D," has an uncertain function but gives rise to the spectrum narrows in algae grown on 2H20 (11, 12). EPR stable EPR signal known as signal II. ZV and D+ have identical experiments on oriented preparations indicate that the inten- EPR spectra and are generally assumed to arise from species sities of the hyperfine couplings follow a 1:3:3:1 pattern (13, with the same chemical structure. Results from a variety of 14), which has led to the suggestion that three protons with experiments have suggested that Z and D are plastoquinones or similar hyperfine coupling constants are present in the Z/D plastoquinone derivatives. In general, however, the evidence to cofactor (13). support this assignment is indirect. To address this situation, From the appearance of the spectrum and its g value, we have developed more direct methods to assign the structure Weaver suggested that Dt is a plastoquinone radical (15). of the ZV/Dt radicals. By selective in vivo deuteration of the Kohl and Wood tested this assignment by extracting methyl groups of plastoquinone in cyanobacteria, we show that plastoquinone from plant chloroplasts and reconstituting the hyperfine couplings from the methyl protons cannot be respon- membranes with deuterated quinone (16). The reconstitution sible for the partially resolved structure seen in the Dt EPR led to a narrowing of the Dt signal and provided strong spectrum. That is, we verify by extraction and mass spectrom- support for Weaver's original proposal. Hales and das Gupta etry that quinones are labeled in algae fed deuterated methio- modeled the EPR spectrum of Dt by using the spin density nine, but no change is observed in the line shape of signal II. distribution of a perturbed plastosemiquinone anion radical Considering the spectral properties ofthe Dt radical, a tyrosine (17). More recently, consideration of the necessarily high origin is a reasonable alternative. In a second series of redox potential of Zt and its unusual spectroscopic proper- experiments, we have found that deuteration of tyrosine does ties led to the identification of the Dt/Zt species as a indeed narrow the Dt signal. Extraction and mass spectral plastoquinone cation radical (18, 19). O'Malley, Babcock, analysis of the quinones in these cultures show that they are not and co-workers attributed the partially resolved hyperfine labeled by tyrosine. These results eliminate a plastoquinone couplings to the 2-methyl group on the PQH2t ring, where PQ origin for Dt; we conclude instead that Dt, and most likely Zt, represents plastoquinone (13, 19). Brok et al. supported the are tyrosine radicals. identification of Z+/Dt species with PQH2t but attributed the hyperfine couplings to a combination of hydroxyl and In plant and algal photosynthesis, photosystem II (PSII) methylene protons (12). Concurrent with the magnetic reso- catalyzes the light-induced oxidation of water and reduction nance studies, Dekker et al. interpreted the Zt minus Z of plastoquinone. The smallest purified unit that is capable of optical absorption spectrum as indicative of a plastoquinone carrying out this reaction consists of seven polypeptides and cation radical (20); Diner and de Vitry obtained similar contains chlorophyll, plastoquinone, manganese, and several optical data but suggested a naphthoquinone cation radical as other bound cofactors (for review, see refs. 1 and 2). Water the likely origin (21). oxidation occurs at a site that contains a cluster of four Mn There are difficulties with the assignment of Z/D to a atoms. This center is interfaced to the photochemically active quinone, however. First, the reconstitution experiments in PSII reaction-center chlorophyll, P680, by at least one ref. 16 were hampered by incomplete extraction of the intermediate electron carrier, which is usually designated original Dt signal and by the necessity of data subtraction to obtain the deuterated Dt spectrum. Second, recent attempts The oxidized form of this cofactor, Zt, has a light-induced to quantify the amount of plastoquinone per reaction center EPR signal that identifies it as an organic radical (3-6). A in PSII preparations indicate that the complex does not variety of kinetic data indicates that Z reduces P680+ directly contain enough quinone to account for Z, D, and the acceptor and that the resulting Zt species is in turn reduced by the quinones; moreover, these samples contain no naphtho- manganese ensemble (refs. 1 and 2, but see refs. 7 and 8). In quinone (22-24). Third, PQH2t has not been a totally addition to the EPR signal from Z+, PSII preparations also successful model for the Zt/D+ EPR spectra. The EPR show a stable EPR signal (signal II) with the same lineshape lineshape of immobilized PQH2t in vitro shows no partially as Zt (9). The radical giving rise to this spectrum is now resolved hyperfine structure (19). In order to reproduce the usually referred to as "D+". Recent data reported by Styring Z+/Dt lineshape, localized chemical perturbations of the and Rutherford suggest that D may be involved in maintain- radical were postulated (13, 19). Similarly, the g values for a ing the integrity of the manganese complex (10). variety of model quinone cation radicals are in the range Because the Zt and Dt EPR spectra are identical, it is 2.0034-2.0038 (25), significantly lower than the 2.0046 value generally assumed that Z and D are species that have the measured for Z+ and Dt. Finally, the lineshape of the D+ radical is independent of temperature over the range 1.5-300 Dt The publication costs of this article were defrayed in part by page charge K (26). If the partially resolved structure in the spectrum payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: PSII, photosystem II; FAB, fast-atom bombardment. 7099 Downloaded by guest on September 23, 2021 7100 Biophysics: Barry and Babcock Proc. Natl. Acad. Sci. USA 84 (1987) were the result of methyl-group hyperfine coupling, a more RESULTS dramatic temperature dependence might be expected (ref. 12; but see ref. 27). In Fig. 1 we present EPR data obtained on the methionine Taken together, these inconsistencies in the Zt/Dt as- auxotroph ofAnabaena grown in the presence of methionine signment suggest that more concrete biochemical and spec- (Fig. 1A) or in the presence of deuterated methionine (Fig. troscopic data on the molecular origin of these signals are 1B). The Dt spectra (solid lines) were recorded in the dark 2 necessary. A definitive assignment could be obtained by mim after illumination. As expected, the Dt spectrum in the deuteration of the species responsible for the ZV/Dt signal control (Fig. 1A) is approximately 20 G wide and has a g value because deuteration will narrow the EPR spectrum (11, 16). of 2.0043. The Dt spectrum in the sample grown on deuter- In bacteria, quinones can be labeled by feeding an amino acid ated methionine (Fig. 1B) is identical in linewidth and g value that is a quinone biosynthetic precursor to a strain that to the control. The major component seen in the light (dashed requires this amino acid for growth (28). This is an attractive spectra) is from the chlorophyll reaction center of photosys- approach to identification of Zt/Dt because the extent of tem I; this signal has a g value of 2.0024. labeling will be high and because in vivo labeling avoids the When mass spectra were recorded on plastoquinone ex- complications of chemical extraction and reconstitution of tracted from these algae, we found the results shown in Fig. membranes. Unfortunately, there are few reports of plant 2. In the mass spectrum of plastoquinone from cultures fed amino acid auxotrophs (29). Instead, we have used cyano- protonated methionine, the molecular ion appears at m/z = bacterial strains in which either quinones or tyrosine can be 749 (Fig. 2A). By contrast, Fig. 2B shows that approximately specifically labeled in vivo. Tyrosine was chosen as a second 48% of the plastoquinone from deuterated cultures was target because the spectral and physical properties of the labeled at both methyl groups, since we found a prominent ZV/Dt species are also consistent with a tyrosine radical. molecular ion at 755. An additional 40% of the quinone was Our EPR results on the specifically deuterated cyanobacteria and the accompanying mass spectral analysis of quinones extracted from these cultures indicate that Dt, and most likely Zt, arise from tyrosine radicals. A 1 MATERIALS AND METHODS A CONTROL The methionine mutant (met-27) of Anabaena variabilis (30) and wild-type Synechocystis 6803 were grown under sterile conditions on BG-11 medium (31); all amino acids were added by sterile filtration. Methionine and the aromatic amino acids were obtained from Sigma. Deuterated methionine, C2H3SC1H2C1H2C1H(N1H2)COO1H (98% 2H3), and deuter- ated tyrosine 1HOC62H4C2H2C2H(NlH2)COO1H (98% 2H7), were from MSD Isotopes (St.