Proc. Natl. Acad. Sci. USAVol. 84, pp. 7099-7103, October 1987Biophysics

Tyrosine radicals are involved in the photosynthetic oxygen-evolving systemBRIDGETTE A. BARRY AND GERALD T. BABCOCKDepartment 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-phyll, at least two other organic cofactors are involved in thephotosynthetic oxygen-evolution process. One of these cofac.tors, called "Z," transfers electrons from the site of wateroxidation to the reaction center of photosystem II. The otherspecies, "D," has an uncertain function but gives rise to thestable EPR signal known as signal II. ZV and D+ have identicalEPR spectra and are generally assumed to arise from specieswith the same chemical structure. Results from a variety ofexperiments have suggested that Z and D are plastoquinones orplastoquinone derivatives. In general, however, the evidence tosupport this assignment is indirect. To address this situation,we have developed more direct methods to assign the structureof the ZV/Dt radicals. By selective in vivo deuteration of themethyl groups of plastoquinone in cyanobacteria, we show thathyperfine couplings from the methyl protons cannot be respon-sible for the partially resolved structure seen in the Dt EPRspectrum. That is, we verify by extraction and mass spectrom-etry that quinones are labeled in algae fed deuterated methio-nine, but no change is observed in the line shape of signal II.Considering the spectral properties ofthe Dt radical, a tyrosineorigin is a reasonable alternative. In a second series ofexperiments, we have found that deuteration of tyrosine doesindeed narrow the Dt signal. Extraction and mass spectralanalysis of the quinones in these cultures show that they are notlabeled by tyrosine. These results eliminate a plastoquinoneorigin for Dt; we conclude instead that Dt, and most likely Zt,are tyrosine radicals.

In plant and algal photosynthesis, photosystem II (PSII)catalyzes the light-induced oxidation of water and reductionof plastoquinone. The smallest purified unit that is capable ofcarrying out this reaction consists of seven polypeptides andcontains chlorophyll, plastoquinone, manganese, and severalother bound cofactors (for review, see refs. 1 and 2). Wateroxidation occurs at a site that contains a cluster of four Mnatoms. This center is interfaced to the photochemically activePSII reaction-center chlorophyll, P680, by at least oneintermediate electron carrier, which is usually designated

The oxidized form of this cofactor, Zt, has a light-inducedEPR signal that identifies it as an organic radical (3-6). Avariety of kinetic data indicates that Z reduces P680+ directlyand that the resulting Zt species is in turn reduced by themanganese ensemble (refs. 1 and 2, but see refs. 7 and 8). Inaddition to the EPR signal from Z+, PSII preparations alsoshow a stable EPR signal (signal II) with the same lineshapeas Zt (9). The radical giving rise to this spectrum is nowusually referred to as "D+". Recent data reported by Styringand Rutherford suggest that D may be involved in maintain-ing the integrity of the manganese complex (10).Because the Zt and Dt EPR spectra are identical, it is

generally assumed that Z and D are species that have the

same chemical structure. This assumption is reinforced bythe distinctive nature of the EPR signal, particularly itspartially resolved hyperfine coupling and its g value of2.0046. The hyperfine structure arises from an interactionbetween the unpaired electron and protons, since the Dtspectrum narrows in algae grown on 2H20 (11, 12). EPRexperiments on oriented preparations indicate that the inten-sities of the hyperfine couplings follow a 1:3:3:1 pattern (13,14), which has led to the suggestion that three protons withsimilar hyperfine coupling constants are present in the Z/Dcofactor (13).From the appearance of the spectrum and its g value,

Weaver suggested that Dt is a plastoquinone radical (15).Kohl and Wood tested this assignment by extractingplastoquinone from plant chloroplasts and reconstituting themembranes with deuterated quinone (16). The reconstitutionled to a narrowing of the Dt signal and provided strongsupport for Weaver's original proposal. Hales and das Guptamodeled the EPR spectrum of Dt by using the spin densitydistribution of a perturbed plastosemiquinone anion radical(17). More recently, consideration of the necessarily highredox potential of Zt and its unusual spectroscopic proper-ties led to the identification of the Dt/Zt species as aplastoquinone cation radical (18, 19). O'Malley, Babcock,and co-workers attributed the partially resolved hyperfinecouplings to the 2-methyl group on the PQH2t ring, where PQrepresents plastoquinone (13, 19). Brok et al. supported theidentification of Z+/Dt species with PQH2t but attributedthe hyperfine couplings to a combination of hydroxyl andmethylene protons (12). Concurrent with the magnetic reso-nance studies, Dekker et al. interpreted the Zt minus Zoptical absorption spectrum as indicative of a plastoquinonecation radical (20); Diner and de Vitry obtained similaroptical data but suggested a naphthoquinone cation radical asthe likely origin (21).There are difficulties with the assignment of Z/D to a

quinone, however. First, the reconstitution experiments inref. 16 were hampered by incomplete extraction of theoriginal Dt signal and by the necessity of data subtraction toobtain the deuterated Dt spectrum. Second, recent attemptsto quantify the amount of plastoquinone per reaction centerin PSII preparations indicate that the complex does notcontain enough quinone to account for Z, D, and the acceptorquinones; moreover, these samples contain no naphtho-quinone (22-24). Third, PQH2t has not been a totallysuccessful model for the Zt/D+ EPR spectra. The EPRlineshape of immobilized PQH2t in vitro shows no partiallyresolved hyperfine structure (19). In order to reproduce theZ+/Dt lineshape, localized chemical perturbations of theradical were postulated (13, 19). Similarly, the g values for avariety of model quinone cation radicals are in the range2.0034-2.0038 (25), significantly lower than the 2.0046 valuemeasured for Z+ and Dt. Finally, the lineshape of the D+radical is independent of temperature over the range 1.5-300K (26). If the partially resolved structure in the Dt spectrum

Abbreviations: PSII, photosystem II; FAB, fast-atom bombardment.

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were the result of methyl-group hyperfine coupling, a moredramatic temperature dependence might be expected (ref. 12;but see ref. 27).Taken together, these inconsistencies in the Zt/Dt as-

signment suggest that more concrete biochemical and spec-troscopic data on the molecular origin of these signals arenecessary. A definitive assignment could be obtained bydeuteration of the species responsible for the ZV/Dt signalbecause deuteration will narrow the EPR spectrum (11, 16).In bacteria, quinones can be labeled by feeding an amino acidthat is a quinone biosynthetic precursor to a strain thatrequires this amino acid for growth (28). This is an attractiveapproach to identification of Zt/Dt because the extent oflabeling will be high and because in vivo labeling avoids thecomplications of chemical extraction and reconstitution ofmembranes. Unfortunately, there are few reports of plantamino acid auxotrophs (29). Instead, we have used cyano-bacterial strains in which either quinones or tyrosine can bespecifically labeled in vivo. Tyrosine was chosen as a secondtarget because the spectral and physical properties of theZV/Dt species are also consistent with a tyrosine radical.Our EPR results on the specifically deuterated cyanobacteriaand the accompanying mass spectral analysis of quinonesextracted from these cultures indicate that Dt, and mostlikely Zt, arise from tyrosine radicals.

MATERIALS AND METHODSThe methionine mutant (met-27) of Anabaena variabilis (30)and wild-type Synechocystis 6803 were grown under sterileconditions on BG-11 medium (31); all amino acids were addedby sterile filtration. Methionine and the aromatic aminoacids 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. Louis). Cells were pelleted forthe EPk studies when the cultures had reached either OD720= 0.22 (Anabaena) or OD720 = 0.44 (Synechocystis). Thepellets were resuspended in buffer containing 7.5% (wt/vol)polyethylene glycol 3400 (Aldrich), 20 mM Hepes (pH 7.5),10 mM CaC12, and 1 mM MgCl2, and the cells were pelletedagain. The final chlorophyll concentration was between 1.0and 1.5 mg/ml for Anabaena and between 0.3 and 0.8 mg/mlfor Synechocystis. EPR spectra were recorded at roomtemperature on a Bruker ER200D spectrometer operating atX-band and using a Varian TM cavity. The power was 10mW, the modulation amplitude was 5 G, the scan time was200 sec, and the time constant was 500 msec. The instru-mentation described (19) was used to determine g values;spin quantitation was carried out as described (32).

After the EPR studies, plastoquinone-9 and the naphtho-quinone, vitamin K1, were isolated from cell cultures by themethod of Schoeder and Lockau (33), except that a C18,uJ3ondapack HPLC column was used. The retention time forvitamin K1 was 18 min and for plastoquinone-9 was 20 min.The absorption spectra of the purified quinones were foundto be identical to those of their respective standards (see alsoref. 34). The plastoquinone standard was a gift from Hof-fman-LaRoche; vitamin K1 was purchased from Sigma.Mass spectra were recorded on a JEOL JMS HX 110 massspectrometer. The sample was ionized by fast-atom bom-bardment (FAB) by using a matrix of triethanolamine and a6-keV (1 eV = 1.602 x 10-19 J) Xenon beam. The acceleratingvoltage was 8 keV, and negative ions were detected. Themolecular weight of plastiquinone is 748 (35). In FAB, theplastoquinone-9 standard gives a strong molecular ion at(M + H)- or 749 and weaker peaks at (M)- and (M - H)-.Similarly, the strongest molecular ion of vitamin K1 appearsat 451. Reduction of the positive and negative ions ofquinones in FAB mass spectrometry has been described (36).

RESULTSIn Fig. 1 we present EPR data obtained on the methionineauxotroph ofAnabaena grown in the presence of methionine(Fig. 1A) or in the presence of deuterated methionine (Fig.1B). The Dt spectra (solid lines) were recorded in the dark 2mim after illumination. As expected, the Dt spectrum in thecontrol (Fig. 1A) is approximately 20 G wide and has a g valueof 2.0043. The Dt spectrum in the sample grown on deuter-ated methionine (Fig. 1B) is identical in linewidth and g valueto the control. The major component seen in the light (dashedspectra) is from the chlorophyll reaction center of photosys-tem I; this signal has a g value of 2.0024.When mass spectra were recorded on plastoquinone ex-

tracted from these algae, we found the results shown in Fig.2. In the mass spectrum of plastoquinone from cultures fedprotonated methionine, the molecular ion appears at m/z =749 (Fig. 2A). By contrast, Fig. 2B shows that approximately48% of the plastoquinone from deuterated cultures waslabeled at both methyl groups, since we found a prominentmolecular ion at 755. An additional 40% of the quinone was

A 1A CONTROL

B DEUTERATEDMETHIONINE

dx

dH

10 G

C DEUTERATEDTYROSINE

g=2.0023

FIG. 1. EPR spectra of cyanobacteria grown in BG-11 mediasupplemented with 90 tLM protonated methionine (A); 90 ,uMdeuterated methionine (B); and 0.5 mM phenylalanine, 0.25 mMtryptophan, and 0.25 mM perdeuterated tyrosine (C). The dashedspectra were recorded in the light; the solid spectra were obtained 2min after illumination. The gain in A is 3.2 x 106, in B is 3.2 x 106,and in C is 5 x 106. Other instrument settings and experimentalconditions are given in Materials and Methods.

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A PROTONATEDMETHONINE A

LLI(-)

z

0

zD

J

LL

B

C

D DEUTERATEDTYROSINE

745 750 755 760 765M/Z

FIG. 2. FAB mass spectra of plastoquinone-9 isolated fromcyanobacteria supplemented with 90 ,uM protonated methionine (A);90 tLM deuterated methionine (B); 0.5 mM phenylalanine, 0.25 mMtryptophan, and 0.25 mM protonated tyrosine (C); and 0.5 mMphenylalanine, 0.25 mM tryptophan, and 0.25 mM perdeuteratedtyrosine (D). In the text the molecular weights are rounded to thenext lower integer (i.e., 749.6 to 749). In A and B, the observed iondistribution from 749 to 753 was reproduced by a computer program(supplied by the manufacturer of the mass spectrometer) that usesthe natural isotope abundance and the molecular formula as inputs.Smaller sample size in C and D makes background peaks moreevident. For example, m/z = 745 is a peak from the triethanolaminematrix.

labeled at one methyl group, with a molecular ion weight of752. Only 12% of the plastoquinone was unlabeled in thesecultures (Table 1). We obtained similar deuterium incorpo-ration results when analyzing the plastoquinone base peak

Table 1. FAB mass spectral data for plastoquinone-9 and forvitamin K1

Relative abundance

Protonated Deuteratedm/z methionine methionine

PQ-9 749 97 12752 3 40755 48

Vitamin K1 451 98 36454 2 64

The quinones were isolated from Anabaena cultures fed eitherprotonated or deuterated methionine. The relative abundance isgiven for each value of m/z. The peak heights were summed, and thevalues were then normalized to give the relative abundance. Thesevalues are approximate, since we must assume that the intensityenvelopes that are centered at each of these m/z values are identical.

(m/z = 189) obtained with electron impact mass spectrom-etry (35) (not shown). In addition to these results onplastoquinone, FAB mass spectra of the naphthoquinone,vitamin K1, shows that this compound was also labeled bydeuterated methionine (Table 1).The EPR and mass spectral data on the methionine

auxotroph show that there was no discernible change in theEPR lineshape of Dt despite extensive deuterium labeling ofthe methyl groups in the quinones. We conclude, therefore,that the methyl groups of quinones are not responsible for thehyperfine couplings observed in the Dt spectrum. In asecond series of experiments, we explored the possibility thata tyrosine radical is responsible for the EPR spectrum.To label tyrosine completely in cyanobacteria, we took

advantage of a feedback inhibition of the aromatic amino acidbiosynthetic pathway that has been well-documented in somespecies (37). A characteristic of such a species is lack ofgrowth in the presence of the inhibiting amino acid and arestoration of growth when all three aromatic amino acids aresupplied (38). Under conditions where phenylalanine, tryp-tophan, and tyrosine are present in the growth medium, thesecultures are dependent on the import of the exogenous aminoacids for growth.

Fig. 3 shows growth curves for a strain with such a patternof sensitivity, Synechocystis 6803. In the presence of 0.5 mMphenylalanine, growth was completely inhibited (Fig. 3B).The addition of tryptophan was not enough to rescue the

1.8

1.6-20~~~~~~~~~

1.4 -

0.2

0.0 -

0.06

0.8 *0/

0.2 b

-j0

0.1 20 40 60 80 100 120 140 160 180 200

TIME (hours)

FIG. 3. Growth curves for Synechocystis 6803 grown in BG-11medium with the following additions: none (e); 0.5 mM phenylala-nine (n); 0.5 mM phenylalanine and 0.25 mM tryptophan (*); and 0.5mM phenylalanine, 0.25 mM tryptophan, and 0.25 mM tyrosine (A).The logarithm of the optical density at 720 nm is plotted versus hoursof culture growth.

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cultures (Fig. 3C), while growth was restored when tyrosine,tryptophan, and phenylalanine were all present in the medi-um (Fig. 3D). Cells that were grown under these conditionswere found to be active in oxygen evolution.

Fig. 1C presents EPR data on Synechocystis grown in thepresence of phenylalanine, tryptophan, and deuterated tyro-sine. The control Dt spectrum (not shown) taken of cellsgrown in the presence of phenylalanine, tryptophan, andprotonated tyrosine was identical to the control spectrum inFig. 1A. In contrast to the 20-G linewidth of the controlsample, we found that the Dt spectrum was narrowed to 7 Gin cultures fed deuterated tyrosine (Fig. 1C). When themodulation amplitude was reduced to 1.4 G, the linewidthdecreased to =6.7 G (not shown). Despite the reduction inlinewidth, however, the g value of the spectrum in thedeuterated tyrosine sample was still 2.0043. Spin quantitationof the Dt signal in the deuterated and protonated tyrosinecultures agreed within several percent, indicating that bothsamples had the same number of Dt radicals, albeit withdifferent line shapes.Mass spectral analysis of plastoquinone, isolated from the

Synechocystis cultures that were fed deuterated tyrosine, ispresented in Fig. 2D. The mass spectrum of plastoquinoneextracted from the algae that were grown on protonatedtyrosine is shown in Fig. 2C. We found no significantincorporation of deuterium into plastoquinone isolated fromthe deuterated cultures. Similarly, the mass spectral analysisof vitamin K1 isolated from the deuterated cultures showedno significant incorporation of label (not shown). Theseresults eliminate a plastoquinone or naphthoquinone originfor Dt. Instead, they provide strong evidence that Dt is atyrosine radical.

DISCUSSION

Although the bulk of the spectroscopic data had favored theidentification of Dt and Z+ as quinone radicals, reliablebiochemical evidence to support this conclusion was lacking.The deuteration approach used by Kohl and Wood (16) is adefinitive way to make the assignment of these cofactors.Since chemical extraction of the membranes and subsequentreconstitution are difficult, however, we have used aminoacid auxotrophs to incorporate deuterium into quinonesbiosynthetically. This strategy is hampered by the lack ofamino acid auxotrophs in plants and green algae, so we haveused cyanobacteria despite the fact that these organisms aremore difficult to deal with biochemically. For example, wehave not been successful in inhibiting 02 evolution in thissystem in a way that allows us to generate and characterizethe deuterated Z+ EPR spectrum. Nonetheless, our resultswith respect to Dt are clear, and we anticipate that theseconclusions will also apply to Z+.The results of our experiments with the Anabaena methio-

nine mutant eliminate a model for Dt in which this cofactoris a quinone radical with a large hyperfine coupling to a ringmethyl group (13). Brok et al. have argued against the methylhyperfine model while maintaining a plastoquinone origin(12); this possibility cannot be discounted from the dataobtained in the methionine experiment. However, the resultsof the tyrosine experiment make all quinone models for Dunlikely. Moreover, these results provide strong positiveevidence for the identification of Dt as a tyrosine radical.First, the mass spectral analysis shows no incorporation oflabel into the benzoquinone, plastoquinone-9 (PQ-A), or thenaphthoquinone, vitamin K1. We assume, therefore, thatother quinones, such as PQ-B and the tocopherol-quinones,

are also unlabeled.* Despite the lack of quinone labeling, thelinewidth of Dt decreases significantly in the deuteratedtyrosine cultures. Second, the amount of deuterium incor-poration into D is high, since the shape of the spectrum andour EPR spin count indicate that all of Dt is labeled. Third,the decrease in the Dt EPR linewidth in this experiment(from 20 G to 7 G) is essentially the same as that observed inthe Dt spectrum when algae are grown on 2H20 (11, 12). Thisobservation suggests that feeding these algae perdeuteratedtyrosine replaces most Dt protons with deuterium, since allprotons with substantial hyperfine couplings must be labeledin order to obtain the 2H20 linewidth. These last two results,the extent of labeling by tyrosine and the extent to which theEPR linewidth is narrowed, are expected if Dt is a tyrosineradical, but are unlikely if Dt is a biosynthetic derivative oftyrosine.There is precedent for the involvement of tyrosine radicals

in biological redox reactions, as this species occurs as anessential component in the enzyme ribonucleotide reductase(44). However, the lineshape of the Zt/Dt EPR spectrumdoes not strongly resemble that of the tyrosine radical inribonucleotide reductase. This lack of similarity may arisefrom sensitivity of the methylene proton hyperfine interac-tions to the dihedral angle that measures their orientationwith respect to the phenol ring (45, 57). In fact, the EPRhyperfine couplings ofEscherichia coli ribonucleotide reduc-tase and of bacteriophage T4 ribonucleotide reductase arequite different; this difference has been accounted for byvariation in this dihedral angle (45). A possible interpretationof the apparent 1:3:3:1 ratio of partially resolved hyperfinelines in the oriented D+ spectrum (13, 14) may involveaccidental degeneracy in the coupling constants of threeprotons, one or two of which are methylene protons. Exper-iments with specifically deuterated tyrosine should enable usto attribute the major hyperfine couplings to particulartyrosine protons.We can address the question of the protonation state of the

Dt tyrosine radical by reference to studies of phenoxyradicals. Neutral and cationic phenoxy radicals are known tohave different g values, with the g value of the neutral,deprotonated radical equal to 2.0044 and that of the proton-ated, cationic species equal to 2.0033 (46). These resultsimply that the Dt radical may be unprotonated. However, thetyrosine radical in ribonucleotide reductase, with a g value of2.0046, is considered to be protonated (47). Proton ENDOR(electron nuclear double resonance) spectroscopy on perdeu-terated Dt will resolve this question because the hydroxylproton, if present, is exchangeable and should be evident inthe spectrum.The assignment of the Z+/Dt species to a tyrosine radical

is consistent with several observations made in other labo-ratories. First, it rationalizes the quinone quantitation data(22-24), since the amount of plastoquinone that is extractedfrom PSII particles is in reasonable agreement with theamount of acceptor quinone that is expected to be present.Second, the optical spectrum of a phenoxy radical (48) bearsa striking resemblance to the absorption difference spectrumof Zt (20). Thus, although the optical data have been used toassign Z to a quinone, they are actually in better agreementwith an assignment of Z/D to a tyrosine. Finally, the redox

*For a review of quinone biosynthesis, see ref. 39. In cyanobacteria,there is a report that plastoquinone can be derived from homogen-tisic acid (40). There is also a study showing that DL-f3-[14C]tyrosineis a precursor of plastoquinone and that methionine is a precursorof plastoquinone and vitamin K1 in Anabaena variabilis (41), inagreement with results on quinone biosynthesis in higher plants (42,43). However, the amount of incorporation of label into the quinonefractions was low in these studies. In any case, only negligibleamounts of quinones other than plastoquinone-9 have been found inPSII preparations (24).

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potentials of phenol and tyrosine radicals are greater than+0.8 V at pH 7 (49, 50). This is in good agreement with theredox potential of Zt, which must be greater than 0.8 V tomediate electron transfer from water to P680. The redoxpotential of the model compounds is also consistent with thehigh redox potential of Dt, which has been reported byBoussac and Etienne (51).A final point concerns the specific tyrosine residues that

give rise to the Zt and Dt radicals. From earlier work, it isclear that these species are associated with the intrinsicmembrane proteins of the PS11 reaction center (32). More-over, other work suggests that D is well shielded from thesolvent (52), which implies that these tyrosine residues maybe associated with membrane-spanning helices. An inspec-tion of the sequences of the D-1 and D-2 polypeptides revealsthat conserved tyrosines occur in the C helices of these twopolypeptides (53-56). These residues are likely candidates forD and Z, since D-1 and D-2 are believed to be the polypep-tides that bind the chlorophyll reaction center (53-56).Site-specific mutagenesis provides a method by which to testthis suggestion.

The authors thank P. Wolk for the gift of the methionine mutant.We are grateful to C. Yocum, R. Debus, and W. Lockau for helpfuldiscussion and to L. McIntosh, S. Ferguson-Miller, and D. McCon-nel for use of their laboratory facilities. We also thank K. Rorrer fortechnical assistance in the maintenance of the cyanobacteria and K.Rehder, E. Oliver, B. Musselman, and J. Stults for recording themass spectra. Mass spectral data were obtained from the MichiganState University Mass Spectrometry Facility supported by a grant(RR-00480) from the Biotechnology Resources Branch, Division ofResearch Resources, National Institutes of Health. B.A.B. is aNational Institutes of Health Postdoctoral Fellow. This work wassupported by the National Institutes of Health (GM 37300) and thePhotosynthesis Program of the Competitive Research Grants Officeof the U.S. Department of Agriculture.

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