Investigation of the differences in the local protein environments surrounding tyrosine radicals YZ • and YD • in photosystem II using wild-type and the D2-tyr160Phe mutant of synechocystis 6803

Xiao Song Tang, Ming Zheng, Dexter A. Chisholm, G Charles Dismukes, Bruce A. Diner

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Abstract

The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ* has been hampered by the simultaneous presence of the much more stable YD , the short lifetime of YZ , and the difficulty in trapping the YZ radical at low temperature. We present here a method for obtaining an uncontaminated YZ radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the β-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ , this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ. We have compared the environments of YZ and YD by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ and YD differ in line shape and line width. (2) Both YZ and YD exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD , indicating the hydrogen bond to be well-defined. In the case of YZ it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 Å. (4) All of the 1H couplings of YD are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ spectrum are broad. The environment surrounding YZ appears to be more disordered and solvent-accessible.

Original languageEnglish
Pages (from-to)1475-1484
Number of pages10
JournalBiochemistry
Volume35
Issue number5
Publication statusPublished - Feb 6 1996

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Synechocystis
Photosystem II Protein Complex
Paramagnetic resonance
Tyrosine
Protons
Hydrogen bonds
Temperature
Hydrogen
Peptides
Proteins
Spectroscopic analysis
Deuterium
Electron Spin Resonance Spectroscopy
Liquid nitrogen
Hydrogen Bonding
Electron Transport
Lighting
Freezing
Linewidth
Labeling

ASJC Scopus subject areas

  • Biochemistry

Cite this

Investigation of the differences in the local protein environments surrounding tyrosine radicals YZ • and YD • in photosystem II using wild-type and the D2-tyr160Phe mutant of synechocystis 6803. / Tang, Xiao Song; Zheng, Ming; Chisholm, Dexter A.; Dismukes, G Charles; Diner, Bruce A.

In: Biochemistry, Vol. 35, No. 5, 06.02.1996, p. 1475-1484.

Research output: Contribution to journalArticle

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title = "Investigation of the differences in the local protein environments surrounding tyrosine radicals YZ • and YD • in photosystem II using wild-type and the D2-tyr160Phe mutant of synechocystis 6803",
abstract = "The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ* has been hampered by the simultaneous presence of the much more stable YD •, the short lifetime of YZ •, and the difficulty in trapping the YZ • radical at low temperature. We present here a method for obtaining an uncontaminated YZ • radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the β-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ • EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ • radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ •, this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ•. We have compared the environments of YZ • and YD • by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ • and YD • differ in line shape and line width. (2) Both YZ • and YD • exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD •, indicating the hydrogen bond to be well-defined. In the case of YZ • it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD • possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 {\AA}. (4) All of the 1H couplings of YD • are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ • spectrum are broad. The environment surrounding YZ • appears to be more disordered and solvent-accessible.",
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T1 - Investigation of the differences in the local protein environments surrounding tyrosine radicals YZ • and YD • in photosystem II using wild-type and the D2-tyr160Phe mutant of synechocystis 6803

AU - Tang, Xiao Song

AU - Zheng, Ming

AU - Chisholm, Dexter A.

AU - Dismukes, G Charles

AU - Diner, Bruce A.

PY - 1996/2/6

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N2 - The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ* has been hampered by the simultaneous presence of the much more stable YD •, the short lifetime of YZ •, and the difficulty in trapping the YZ • radical at low temperature. We present here a method for obtaining an uncontaminated YZ • radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the β-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ • EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ • radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ •, this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ•. We have compared the environments of YZ • and YD • by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ • and YD • differ in line shape and line width. (2) Both YZ • and YD • exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD •, indicating the hydrogen bond to be well-defined. In the case of YZ • it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD • possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 Å. (4) All of the 1H couplings of YD • are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ • spectrum are broad. The environment surrounding YZ • appears to be more disordered and solvent-accessible.

AB - The reaction center of photosystem II (PSII) of the oxygenic photosynthetic electron transport chain contains two redox-active tyrosines, Tyr160 (YD) of the D2 polypeptide and Tyr161 (YZ) of the D1 polypeptide, each of which may be oxidized by the primary electron donor, P680+. Spectroscopic characterization of YZ* has been hampered by the simultaneous presence of the much more stable YD •, the short lifetime of YZ •, and the difficulty in trapping the YZ • radical at low temperature. We present here a method for obtaining an uncontaminated YZ • radical, trapped by freezing under illumination of PSII core complexes isolated from YD-less mutants of Synechocystis 6803. Specific labeling with deuterium of the β-methylene-3,3- or of the ring 3,5-protons of the PSII reaction center tyrosines in the YD-less D2-Tyr160Phe mutant results in a change in the hyperfine structure of the YZ • EPR signal, further confirming that this signal indeed arises from tyrosine. The trapped YZ • radical is also stable for several months at liquid nitrogen temperature. Due to both the absence of contaminating paramagnetic species and the stability at low temperature of YZ •, this mutant core complex constitutes an excellent experimental system for the spectroscopic analysis of YZ•. We have compared the environments of YZ • and YD • by EPR, 1H ENDOR, and TRIPLE spectroscopies using both mutant and wild-type core complexes, with the following observations: (1) the EPR spectra of YZ • and YD • differ in line shape and line width. (2) Both YZ • and YD • exhibit D2O-exchangeable 1H hyperfine coupling near 3 MHz, consistent with the presence of a hydrogen bond from a proton donor to the phenolic oxygen atom of a neutral tyrosyl radical. This hyperfine coupling is sharp in the case of YD •, indicating the hydrogen bond to be well-defined. In the case of YZ • it is broad, suggestive of a distribution of hydrogen-bonding distances. (3) YD • possesses three additional weak couplings that disappear in D2O, arising from three or fewer protons (protein or solvent) located within a shell between 4.5 and 8.5 Å. (4) All of the 1H couplings of YD • are sharp, which is indicative of a well-ordered protein environment. (5) All of the 1H couplings in the YZ • spectrum are broad. The environment surrounding YZ • appears to be more disordered and solvent-accessible.

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