FOURIER-TRANSFORM INFRARED DIFFERENCE SPECTROSCOPY OF PHOTOSYSTEM-II TYROSINE-D USING SITE-DIRECTED MUTAGENESIS AND SPECIFIC ISOTOPE LABELING

Tyrosine D (Tyr(D)), a side path electron carrier of photosystem II (P S II), has been studied by light-induced Fourier transform infrared (F TIR) difference spectroscopy in PS II care complexes of Synechocystis sp. PCC 6803 using the experimental conditions previously optimized to generate the pure TyrD(.)/Tyr(D) FTIR difference spectrum in PS II-en riched membranes of spinach [Hienerwadel, R., Boussac, A., Breton, J., and Berthomieu, C. (1996) Biochemistry 35, 115447-115460]. IR modes o f Tyr(D) and Tyr(D)(.) have been identified by specific H-2- or C-13-I abeling of the tyrosine side chains. The v(8a)(CC) and v(19)(CC) IR mo des of Tyro are identified at 1615 and 1513-1510 cm(-1), respectively, These frequencies show that Tyro is protonated. Comparison of isotope -sensitive signals in situ with those of the model compound p-methylph enol dissolved in different solvents leads to the assignment of the v( 7'a)(CO) and delta(COH) modes of Tyro at 1275 and 1250 cm(-1), respect ively. It is shown that these modes and in particular the delta(COH) I R mode are very sensitive to the formation of hydrogen-bonded complexe s with amide C=O or with imidazole nitrogen atoms. The frequencies obs erved in situ show that Tyr(D) is hydrogen-bonded to the imidazole rin g of a neutral histidine. For the radical Tyr(D)(.), isotope-sensitive IX modes are identified at 1532 and 1503 cm(-1). The signal at 1503 c m(-1) is assigned to the v(CO) mode of Tyr(D)(.) since it is sensitive to C-13-labeling at the ring carbon involved in the C4-O bond. The pe rturbation of Tyr(D) and Tyr(D)(.) IR modes upon site-directed replace ment of D2-His 189 by Gin confirms that a hydrogen bond exists between both Tyr(D) and Tyr(D)(.) and D2-His189. In the D20-His189Gln mutant, the v(7'a)(CO) mode of Tyro at 1267 cm(-1) and the delta(COH) mode at approximate to 1228 cm(-1) show that a hydrogen bond is formed betwee n Tyr(D) and an amide carbonyl, probably that of the D2-Gln189 side ch ain. Electron nuclear double resonance (ENDOR) measurements have shown that Tyr(D)(.) is hydrogen-bonded in the wild type but not in the mut ant [Tang, X.-S., Chrisholm, D, A., Dismukes, G. C., Brudwig, G. W., a nd Diner, B. A. (1993) Biochemistry 32, 13742-13748]. The v(CO) mode o f Tyr(D)(.) at 1497 cm(-1) is downshifted by 6 cm(-1) compared To WT P S]I, indicating that hydrogen bonding induces a frequency upshift of t he v(CO) IR mode of Tyr(.), IR signals from the Gin side chain v(C=O) mode are proposed to contribute at 1659 and 1692 cm(-1) in the Tyr(D) and Tyr(D)(.) states, respectively, These frequencies are consistent w ith the rupture of a hydrogen bond upon Tyr(D)(.) formation in the mut ant. The frequency of the v(CO) mode of Tyr(D)(.), observed al 1503 cm (-1) for WT PS II, is intermediate between that observed at 1497 cm(-1 ) in the D2-Wis189Gln mutant and at 1513 cm(-1) for Tyr(.) formed by U V irradiation in berate buffer, suggesting weaker or fewer hydrogen bo nds for Tyr(D)(.) in PS II than in solution. The role of D2-His189 in proton uptake upon TyrD(.) formation is also investigated.