Structural changes in chromatophores of Rhodobacter sphaeroides reaction center mutants associated with the substitution of amino acid residues near the primary electron donor P have been investigated by light-induced FTIR difference spectroscopy. The single-site mutations Leu-L131 to His and Leu-M160 to His and the corresponding double mutation were designed to introduce a proton-donating residue that could form a hydrogen bond with the keto carbonyl of ring V of each bacteriochlorophyll (PL and PM) of the dimer. The presence of large positive bands at ≈1550, 1480, and 1295 cm−1, as well as at 2600–2800 cm−1 in the light-induced P+QA−/PQA FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone QA, demonstrates that the BChl dimer state of P+ is preserved in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, although frequency shifts and amplitude changes can be observed, notably for LH(M160). Compared to wild type, these changes are thought to reflect a different charge repartition over the two BChls in P+. Large frequency downshifts in the 9-keto CO stretching region of the P+QA−/PQA FTIR difference spectra of chromatophores are observed in the mutant samples relative to wild type. For the LH(M160) mutant, a large differential signal at 1678/1664 cm−1 is assigned to a shift, upon photooxidation, of the 9-keto CO of PM hydrogen-bonded to His-M160, while that at 1718/1696 cm−1 corresponds to the free 9-keto CO of PL. For the LH(L131) mutant, a signal at 1657 cm−1 is assigned to the 9-keto CO of PL hydrogen-bonded to His-L131 while two signals at 1692 and 1682 cm−1 are possible candidates for PL+. For the double mutant, the main differential signal at 1685/1662 cm−1, which is downshifted by ≈20 cm−1 with respect to wild type, is interpreted in terms of the superposition of the contributions from the 9-keto CO of both PL and PM hydrogen-bonded to His-L131 and His-M160, respectively. The changes observed in the IR spectra of the mutants support the conclusion that a hydrogen bond has been introduced to the dimer at the 9-keto CO of PL and/or PM, and they suggest a stronger hydrogen bond in LH(L131) than in LH(M160). From the present data and those previously reported for heterodimer mutants where one bacteriochlorophyll PL or PM is replaced by bacteriopheophytin [Nabedryk, e., Robles, S. j., Goldman, e., Youvan, D. C., & Breton, J. (1992) Biochemistry 31, 10852–10858], a clear identification of the vibrational modes of PL and PM in the wild-type reaction center can be made. The band at 1683 cm−1 and the shoulder at 1692 cm−1 are assigned to the 9-keto CO vibrations of PM and PL, respectively, their counterparts appearing at 1705 and 1713 cm−1 in the photooxidized state.
ASJC Scopus subject areas