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have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine.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 language | English |
|---|---|
| Pages (from-to) | 6960-6969 |
| Number of pages | 10 |
| Journal | Biochemistry |
| Volume | 28 |
| Issue number | 17 |
| Publication status | Published - 1989 |
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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 journal › Article
}
TY - JOUR
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
Y1 - 1989
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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UR - http://www.scopus.com/inward/citedby.url?scp=0024976548&partnerID=8YFLogxK
M3 - Article
C2 - 2510819
AN - SCOPUS:0024976548
VL - 28
SP - 6960
EP - 6969
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 17
ER -