Characterization of the Interaction between Manganese and Tyrosine Z in Acetate-Inhibited Photosystem II ² Veronika A. Szalai, ‡ Henriette Ku ¨hne, § K. V. Lakshmi, ‡ and Gary W. Brudvig* ,‡ Department of Chemistry and Department of Molecular Biophysics and Biochemistry, Yale UniVersity, New HaVen, Connecticut 06520 ReceiVed June 1, 1998; ReVised Manuscript ReceiVed July 28, 1998 ABSTRACT: When acetate-inhibited photosystem II (PSII) membranes are illuminated at temperatures above 250 K and quickly cooled to 77 K, a 240 G-wide electron paramagnetic resonance (EPR) signal is observed at 10 K. This EPR signal arises from a reciprocal interaction between the spin 1 / 2 ground state of the S 2 state of the Mn 4 cluster, for which a multiline EPR signal with shifted 55 Mn hyperfine peaks is observed, and the oxidized tyrosine residue, Y Z • , for which a broadened Y Z • EPR spectrum is observed. The S 2 Y Z • EPR signal in acetate-inhibited PSII is the first in which characteristic spectral features from both paramagnets can be observed. The observation of distinct EPR signals from each of the paramagnets together with the lack of a half-field EPR transition indicates that the exchange and dipolar couplings are weak. Below 20 K, the S 2 Y Z • EPR signal in acetate-inhibited PSII is in the static limit. Above 20 K, the line width narrows dramatically as the broad low-temperature S 2 Y Z • EPR signal is converted to a narrow Y Z • EPR signal at room temperature. The line width narrowing is interpreted to be due to averaging of the exchange and dipolar interactions between Y Z • and the S 2 state of the Mn 4 cluster by rapid spin- lattice relaxation of the Mn 4 cluster as the temperature is increased. Decay of the S 2 Y Z • intermediate at 200 K shows that the g ) 4.1 form of the S 2 state is formed and that a noninteracting S 2 -state multiline EPR signal is not observed as an intermediate in the decay. This result shows that a change in the redox state of Y Z induces a spin-state change in the Mn 4 cluster in acetate-inhibited PSII. The interconversion between spin states of the Mn 4 cluster in acetate-inhibited PSII supports the idea that Y Z oxidation or Y Z • reduction is communicated to the Mn 4 cluster through a direct hydrogen-bonding pathway, possibly involving a ligand bound to the Mn 4 cluster. In green plants, light-driven oxidation of water to dioxygen and reduction of plastoquinone to plastoquinol are carried out by the membrane-bound protein complex photosystem II (PSII) 1 (1-3). To produce dioxygen, the O 2 -evolving complex (OEC), containing a tetranuclear manganese-oxo cluster, is oxidized in four photochemical steps and cycles through five “store” or S states labeled S 0 -S 4 (4). On the basis of X-ray absorption measurements of the dark-stable S 1 state, the Mn 4 cluster has been modeled as an unsym- metrical dimer of dimers (5), although the structure of the Mn 4 cluster is still under debate. The S 2 state is an odd- electron state, and electron paramagnetic resonance (EPR) spectroscopy has been used extensively to study the Mn 4 cluster in the S 2 state [reviewed in Miller and Brudvig (6)]. The physiological oxidant of the Mn 4 cluster is a redox- active tyrosine known as Y Z (7, 8). In O 2 -evolving PSII membranes, oxidized Y Z is difficult to observe by EPR spectroscopy because its re-reduction by the Mn 4 cluster is extremely rapid. However, time-resolved EPR has been used to observe Y Z • at room temperature in O 2 -evolving PSII samples (9, 10). Y Z • can also be trapped by freezing a Mn- depleted PSII sample under illumination (11). In both samples, Y Z • exhibits an EPR signal less than 30 G wide that is similar to the EPR signal of the dark-stable tyrosine radical in PSII, Y D • . Several chemical treatments, including depletion of calcium or chloride or addition of acetate, ammonia, or fluoride, block the S-state cycle and cause a shutdown of the water oxidation chemistry at the S 2 state. These inhibitory treatments may perturb the binding of substrate water or displace chloride or calcium ions required for maximal O 2 evolution. When such inhibited PSII samples are continuously illuminated at temperatures above 250 K and then quickly cooled to 77 K, an EPR signal greater than 100 G wide and centered at g ) 2 can be observed at 10 K (12-20). This signal, previously referred to as the S3 EPR signal, has been identified as arising from the interaction of Y Z • (21, 22) with the S 2 state of the Mn 4 cluster in acetate- inhibited PSII samples (23). However, during steady-state illumination at room temperature, a narrow Y Z • EPR signal, similar to that observed in Mn-depleted PSII samples, has ² Supported by the National Institutes of Health (Grants GM 32715 and GM 36442) and by a National Institutes of Health predoctoral traineeship to V.A.S. (GM 08283). * To whom correspondence should be addressed. Telephone: (203) 432-5202. Fax: (203) 432-6144. E-mail: Gary.Brudvig@yale.edu. ‡ Department of Chemistry. § Department of Molecular Biophysics and Biochemistry. 1 Abbreviations: chl, chlorophyll; DCMU, 3-(3,4-dichlorophenyl)- 1,1-dimethylurea; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol bis(-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; EPR, electron para- magnetic resonance; MES, 2-morpholinoethanesulfonic acid; NO • , nitric oxide; OEC, O2-evolving complex; PPBQ, phenyl-p-benzoquinone; PSII, photosystem II; YD, tyrosine 160 of the D2 polypeptide; YZ, tyrosine 161 of the D1 polypeptide. 13594 Biochemistry 1998, 37, 13594-13603 S0006-2960(98)01302-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/05/1998