Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131

E. Nabedryk, James Paul Allen, A. K W Taguchi, J. C. Williams, N. W. Woodbury, J. Breton

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Abstract

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 (P(L) and P(M)) 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+Q(A)-/PQ(A) FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone Q(A), 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 C=O stretching region of the P+Q(A)- /PQ(A) 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 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, 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 C=O of both P(L) and P(M) 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 C=O of P(L) and/or P(M), 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 P(L) or P(M) is replaced by bacteriopheophytin [Nabedryk, E., Robles, S. J., Goldman, E., Youvan, D. C., and Breton, J. (1992) Biochemistry 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 appearing at 1705 and 1713 cm-1 in the photooxidized state.

Original languageEnglish
Pages (from-to)13879-13885
Number of pages7
JournalBiochemistry
Volume32
Issue number50
DOIs
Publication statusPublished - 1993

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Chromatophores
Rhodobacter sphaeroides
Fourier Analysis
Dimers
Bacteriochlorophylls
Hydrogen
Fourier transforms
Hydrogen bonds
Photooxidation
Electrons
Infrared radiation
Fourier Transform Infrared Spectroscopy
Biochemistry
Stretching
Protons
Substitution reactions
Spectroscopy
Light
Amino Acids
Mutation

ASJC Scopus subject areas

  • Biochemistry

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Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131. / Nabedryk, E.; Allen, James Paul; Taguchi, A. K W; Williams, J. C.; Woodbury, N. W.; Breton, J.

In: Biochemistry, Vol. 32, No. 50, 1993, p. 13879-13885.

Research output: Contribution to journalArticle

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title = "Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131",
abstract = "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 (P(L) and P(M)) 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+Q(A)-/PQ(A) FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone Q(A), 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 C=O stretching region of the P+Q(A)- /PQ(A) 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 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, 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 C=O of both P(L) and P(M) 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 C=O of P(L) and/or P(M), 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 P(L) or P(M) is replaced by bacteriopheophytin [Nabedryk, E., Robles, S. J., Goldman, E., Youvan, D. C., and Breton, J. (1992) Biochemistry 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 appearing at 1705 and 1713 cm-1 in the photooxidized state.",
author = "E. Nabedryk and Allen, {James Paul} and Taguchi, {A. K W} and Williams, {J. C.} and Woodbury, {N. W.} and J. Breton",
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TY - JOUR

T1 - Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131

AU - Nabedryk, E.

AU - Allen, James Paul

AU - Taguchi, A. K W

AU - Williams, J. C.

AU - Woodbury, N. W.

AU - Breton, J.

PY - 1993

Y1 - 1993

N2 - 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 (P(L) and P(M)) 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+Q(A)-/PQ(A) FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone Q(A), 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 C=O stretching region of the P+Q(A)- /PQ(A) 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 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, 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 C=O of both P(L) and P(M) 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 C=O of P(L) and/or P(M), 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 P(L) or P(M) is replaced by bacteriopheophytin [Nabedryk, E., Robles, S. J., Goldman, E., Youvan, D. C., and Breton, J. (1992) Biochemistry 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 appearing at 1705 and 1713 cm-1 in the photooxidized state.

AB - 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 (P(L) and P(M)) 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+Q(A)-/PQ(A) FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone Q(A), 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 C=O stretching region of the P+Q(A)- /PQ(A) 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 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, 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 C=O of both P(L) and P(M) 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 C=O of P(L) and/or P(M), 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 P(L) or P(M) is replaced by bacteriopheophytin [Nabedryk, E., Robles, S. J., Goldman, E., Youvan, D. C., and Breton, J. (1992) Biochemistry 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 appearing at 1705 and 1713 cm-1 in the photooxidized state.

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