FOURIER-TRANSFORM INFRARED STUDY OF THE PRIMARY ELECTRON-DONOR IN CHROMATOPHORES OF RHODOBACTER-SPHAEROIDES WITH REACTION CENTERS GENETICALLY-MODIFIED AT RESIDUE-M160 AND RESIDUE-L131

Structural changes in chromatophores of Rhodobacter sphaeroides reacti on center mutants associated with the substitution of amino acid resid ues near the primary electron donor P have been investigated by light- induced FTIR difference spectroscopy. The single-site mutations Leu-L1 31 to His and Leu-M160 to His and the corresponding double mutation we re designed to introduce a proton-donating residue that could form a h ydrogen bond with the keto carbonyl of ring V of each bacteriochloroph yll (P(L) and P(M)) of the dimer. The presence of large positive bands at almost-equal-to 1550, 1480, and 1295 cm-1, as well as at 2600-2800 cm-1 in the light-induced P+Q(A)-/PQ(A) FTIR difference spectra, corr esponding to the photooxidation of P and the photoreduction of the pri mary quinone Q(A), demonstrates that the BChl dimer state of P+ is pre served in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, altho ugh frequency shifts and amplitude changes can be observed, notably fo r LH(MI60). Compared to wild type, these changes are thought to reflec t a different charge repartition over the two BChls in P+. Large frequ ency downshifts in the 9-keto C=O stretching region of the P+Q(A)-/PQ( A) FTIR difference spectra of chromatophores are observed in the mutan t samples relative to wild type. For the LH(M160) mutant, a large diff erential signal at 1678/1664 cm-1 is assigned to a shift, upon photoox idation, of the 9-keto C=O of P(M) hydrogen-bonded to His-M160, while that at 1718/1696 cm-1 corresponds to the free 9-keto C=O Of P(L). For the LH(L131) mutant, a signal at 1657 cm-1 is assigned to the 9-keto C=O of P(L) hydrogen-bonded to His-L131 while two signals at 1692 and 1682 cm-1 are possible candidates for P(L)+. For the double mutant, th e main differential signal at 1685/1662 cm-1, which is downshifted by almost-equal-to 20 cm-1 with respect to wild type, is interpreted in t erms of the superposition of the contributions from the 9-keto C=O of both P(L) and P(M) hydrogen-bonded to His-L131 and His-M160, respectiv ely. The changes observed in the IR spectra of the mutants support the conclusion that a hydrogen bond has been introduced to the dimer at t he 9-keto C=O Of P(L) and/or P(M), and they suggest a stronger hydroge n bond in LH(L131) than in LH(M160). From the present data and those p reviously reported for heterodimer mutants where one bacteriochlorophy ll P(L) or P(M) is replaced by bacteriopheophytin [Nabedryk, E., Roble s, S. J., Goldmar., E., Youvan, D. C., & Breton, J. (1992) Biochemistr y 31, 10852-10858], a clear identification of the vibrational modes of P(L) and P(M) 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 C=O vibrations of P(M) and P(L), respectively, their counterparts appe aring at 1705 and 1713 cm-1 in the photooxidized state.