Proton Uptake by Carboxylic Acid Groups upon Photoreduction of the Secondary Quinone (Q_B) in Bacterial Reaction Centers from Rhodobacter sphaeroides: FTIR Studies on the Effects of Replacing Glu H173

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Abstract

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, Glu H173, located approximately 7 A from the center of the secondary quinone acceptor QB, is expected to contribute to proton uptake upon QB- formation in response to the movement of an electron in its vicinity. Steady-state FTIR difference spectroscopy provides a method to monitor proton uptake by carboxylic acids upon photochemical changes. The FTIR spectra corresponding to the photoreduction of QB were obtained at pH 7 for RCs containing Glu (native), Gln (EQ H173), or Asp (ED H173) at the H173 site. No new bands were observed in the carboxylic acid region (1770-1700 cm-1) in any of the mutant RCs compared to native RCs. In addition, the positive band at 1728 cm-1, previously assigned to Glu L212 [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., Mäntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732], remained present in all of the mutant RCs. This result shows that Glu H173 is not a major contributor to proton uptake upon QB- formation and further strengthens the assignment of the 1728 cm-1 band to Glu L212. An increase in the 1728 cm-1 band was observed in the EQ H173 RCs compared to that of either the ED H173 or native RCs. These changes are consistent with Glu and Asp at H173 remaining ionized in the QB and QB- states. Changes in the absorption regions of the semiquinone and amide or side chain groups in the spectra of the mutant RCs suggest slight changes in the protein structure compared to those of native RCs, which could contribute to the altered kinetics observed in the mutant RCs.

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