It is demonstrated by ENDOR and Special TRIPLE spectroscopy that two distinct radical anion states of the intermediate electron acceptor (I), a bacteriopheophytin, can be freeze-trapped in isolated photosynthetic reaction centers of Rhodobacter sphaeroides. The formation of these states depends on the illumination time prior to freezing and the temperature. The first state, I1.-, is metastable and relaxes irreversibly at T ≃ 160 K to the second state, I2.-. Experiments on quinone depleted as well as mutant: reaction centers help to exclude the possibility that other cofactors besides the bacteriopheophytin in the A-branch, Φ(A), are reduced during the trapping procedure. In particular, two mutants are investigated, in which the hydrogen bonds to Φ(A) that exist in the wild type are removed. These mutants are EL(L104), in which Glu at position L104 near the 131-keto group of Φ(A) is replaced by Leu, and WF(L100), in which Trp at position L100 near the 132- methyl ester of Φ(A) is replaced by Phe. Both mutations have characteristic effects on both I.- states. In addition, the replacement of Thr at position M133 near the 131-keto group of the inactive bacteriopheophytin and of Gly at position M203 near the 131-keto group of the accessory bacteriochlorophyll in the A-branch by Asp causes no changes of the electronic structure of I.-. The two I.- states are interpreted in terms of a reorientation of the 3-acetyl group of Φ(A) after reduction. Possible implications for the initial charge separation process are discussed.
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