Directed alteration of the D1 polypeptide of photosystem II

Evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680

James G. Metz, Peter J. Nixon, Matthias Rögner, Gary W Brudvig, Bruce A. Diner

Research output: Contribution to journalArticle

268 Citations (Scopus)

Abstract

In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA - occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA - occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.

Original languageEnglish
Pages (from-to)6960-6969
Number of pages10
JournalBiochemistry
Volume28
Issue number17
Publication statusPublished - 1989

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Photosystem II Protein Complex
Oxidation-Reduction
Tyrosine
Microbial Drug Resistance
Genes
Electrons
Diuron
Oxygen
Synechocystis
Peptides
Chlorophyll
Anti-Bacterial Agents
Genetic Recombination
Genetic Complementation Test
Hydroxylamine
Cyanobacteria
Phenylalanine
DNA Sequence Analysis
Amplification
Paramagnetic resonance

ASJC Scopus subject areas

  • Biochemistry

Cite this

Directed alteration of the D1 polypeptide of photosystem II : Evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680. / Metz, James G.; Nixon, Peter J.; Rögner, Matthias; Brudvig, Gary W; Diner, Bruce A.

In: Biochemistry, Vol. 28, No. 17, 1989, p. 6960-6969.

Research output: Contribution to journalArticle

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title = "Directed alteration of the D1 polypeptide of photosystem II: Evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680",
abstract = "In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA - occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA - occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.",
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T1 - Directed alteration of the D1 polypeptide of photosystem II

T2 - Evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680

AU - Metz, James G.

AU - Nixon, Peter J.

AU - Rögner, Matthias

AU - Brudvig, Gary W

AU - Diner, Bruce A.

PY - 1989

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N2 - In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA - occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA - occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.

AB - In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R. J., Barry, B. A., Babcock, G. T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA - occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA - occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.

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