Resolution and reconstitution of the cyanobacterial photosystem I complex

Kevin G. Parrett, Tetemke Mehari, John H. Golbeck

Research output: Contribution to journalArticle

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Abstract

We had shown previously that addition of urea to a Synechococcus 6301 Photosystem I complex leads to dissociation of the 8.9 kDa, FA FB polypeptide, from the P-700- and FX-containing Photosystem I core protein (Golbeck et al. (1988) FEBS Lett. 240, 9-14). In the presence of chaotropes, the iron-sulfur clusters in the 8.9 kDa, FA FB polypeptide are unstable, and degrade to the level of zero-valence sulfur (Parrett et al. (1989) Biochim. Biophys. Acta 973, 324-332). We now report that addition of FeCl3, Na2S, and β-mercaptoethanol to a mixture of the low molecular mass polypeptides and the purified Photosystem I core protein results in complete restoration of light-induced charge separation between P-700 and FA FB, including (i) the 30 ms room temperature charge recombination between P-700+ and [ FA FB]- and (ii) the characteristic light-induced ESR spectrum of FA and FB with g values of 2.05, 1.94, 1.92 and 1.89. Analysis by SDS-PAGE shows that the reconstituted 8.9 kDa, FA FB polypeptide has rebound to the Photosystem I core protein. The purified Photosystem I core protein was treated with 3 M urea and 5 mM potassium ferricyanide to oxidatively denature FX to the level of zero-valence sulfur; light-induced charge separation in the apo-FX core protein results in a 3 μs optical transient due to the relaxation of the P-700 triplet state. Addition of FeCl3, Na2S and β-mercaptoethanol results in restoration of light-induced charge separation between P-700 and FX, including (i) the 1.2 ms room temperature charge recombination between P-700+ and FX - and (ii) the characteristic light-induced ESR resonances of FX with g values of 2.05, 1.86 and 1.78. Addition of FeCl3, Na2S and β-mercaptoethanol to a mixture of the FX and FA FB apoproteins results in reconstitution of electron flow from P-700 to FA FB, indicating quantitative reinsertion of the FX as well as the FA FB iron-sulfur clusters and quantitative rebinding of the 8.9 kDa polypeptide to the Photosystem I core protein. This reconstitution technique makes possible novel studies of Photosystem I, including chemical or genetic modification of the FX or FA FB apoproteins followed by reinsertion of the iron-sulfur clusters and rebinding of the low molecular mass polypeptides to produce a functional Photosystem I complex.

Original languageEnglish (US)
Pages (from-to)341-352
Number of pages12
JournalBBA - Bioenergetics
Volume1015
Issue number2
DOIs
StatePublished - Feb 2 1990

Fingerprint

Photosystem I Protein Complex
Sulfur
Peptides
Mercaptoethanol
Light
Proteins
Apoproteins
Iron
Molecular mass
Genetic Recombination
Restoration
Paramagnetic resonance
Urea
Synechococcus
Temperature
Polypeptides
chlorophyll P 700
Polyacrylamide Gel Electrophoresis
Electrons

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

Parrett, Kevin G. ; Mehari, Tetemke ; Golbeck, John H. / Resolution and reconstitution of the cyanobacterial photosystem I complex. In: BBA - Bioenergetics. 1990 ; Vol. 1015, No. 2. pp. 341-352.
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abstract = "We had shown previously that addition of urea to a Synechococcus 6301 Photosystem I complex leads to dissociation of the 8.9 kDa, FA FB polypeptide, from the P-700- and FX-containing Photosystem I core protein (Golbeck et al. (1988) FEBS Lett. 240, 9-14). In the presence of chaotropes, the iron-sulfur clusters in the 8.9 kDa, FA FB polypeptide are unstable, and degrade to the level of zero-valence sulfur (Parrett et al. (1989) Biochim. Biophys. Acta 973, 324-332). We now report that addition of FeCl3, Na2S, and β-mercaptoethanol to a mixture of the low molecular mass polypeptides and the purified Photosystem I core protein results in complete restoration of light-induced charge separation between P-700 and FA FB, including (i) the 30 ms room temperature charge recombination between P-700+ and [ FA FB]- and (ii) the characteristic light-induced ESR spectrum of FA and FB with g values of 2.05, 1.94, 1.92 and 1.89. Analysis by SDS-PAGE shows that the reconstituted 8.9 kDa, FA FB polypeptide has rebound to the Photosystem I core protein. The purified Photosystem I core protein was treated with 3 M urea and 5 mM potassium ferricyanide to oxidatively denature FX to the level of zero-valence sulfur; light-induced charge separation in the apo-FX core protein results in a 3 μs optical transient due to the relaxation of the P-700 triplet state. Addition of FeCl3, Na2S and β-mercaptoethanol results in restoration of light-induced charge separation between P-700 and FX, including (i) the 1.2 ms room temperature charge recombination between P-700+ and FX - and (ii) the characteristic light-induced ESR resonances of FX with g values of 2.05, 1.86 and 1.78. Addition of FeCl3, Na2S and β-mercaptoethanol to a mixture of the FX and FA FB apoproteins results in reconstitution of electron flow from P-700 to FA FB, indicating quantitative reinsertion of the FX as well as the FA FB iron-sulfur clusters and quantitative rebinding of the 8.9 kDa polypeptide to the Photosystem I core protein. This reconstitution technique makes possible novel studies of Photosystem I, including chemical or genetic modification of the FX or FA FB apoproteins followed by reinsertion of the iron-sulfur clusters and rebinding of the low molecular mass polypeptides to produce a functional Photosystem I complex.",
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Resolution and reconstitution of the cyanobacterial photosystem I complex. / Parrett, Kevin G.; Mehari, Tetemke; Golbeck, John H.

In: BBA - Bioenergetics, Vol. 1015, No. 2, 02.02.1990, p. 341-352.

Research output: Contribution to journalArticle

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T1 - Resolution and reconstitution of the cyanobacterial photosystem I complex

AU - Parrett, Kevin G.

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N2 - We had shown previously that addition of urea to a Synechococcus 6301 Photosystem I complex leads to dissociation of the 8.9 kDa, FA FB polypeptide, from the P-700- and FX-containing Photosystem I core protein (Golbeck et al. (1988) FEBS Lett. 240, 9-14). In the presence of chaotropes, the iron-sulfur clusters in the 8.9 kDa, FA FB polypeptide are unstable, and degrade to the level of zero-valence sulfur (Parrett et al. (1989) Biochim. Biophys. Acta 973, 324-332). We now report that addition of FeCl3, Na2S, and β-mercaptoethanol to a mixture of the low molecular mass polypeptides and the purified Photosystem I core protein results in complete restoration of light-induced charge separation between P-700 and FA FB, including (i) the 30 ms room temperature charge recombination between P-700+ and [ FA FB]- and (ii) the characteristic light-induced ESR spectrum of FA and FB with g values of 2.05, 1.94, 1.92 and 1.89. Analysis by SDS-PAGE shows that the reconstituted 8.9 kDa, FA FB polypeptide has rebound to the Photosystem I core protein. The purified Photosystem I core protein was treated with 3 M urea and 5 mM potassium ferricyanide to oxidatively denature FX to the level of zero-valence sulfur; light-induced charge separation in the apo-FX core protein results in a 3 μs optical transient due to the relaxation of the P-700 triplet state. Addition of FeCl3, Na2S and β-mercaptoethanol results in restoration of light-induced charge separation between P-700 and FX, including (i) the 1.2 ms room temperature charge recombination between P-700+ and FX - and (ii) the characteristic light-induced ESR resonances of FX with g values of 2.05, 1.86 and 1.78. Addition of FeCl3, Na2S and β-mercaptoethanol to a mixture of the FX and FA FB apoproteins results in reconstitution of electron flow from P-700 to FA FB, indicating quantitative reinsertion of the FX as well as the FA FB iron-sulfur clusters and quantitative rebinding of the 8.9 kDa polypeptide to the Photosystem I core protein. This reconstitution technique makes possible novel studies of Photosystem I, including chemical or genetic modification of the FX or FA FB apoproteins followed by reinsertion of the iron-sulfur clusters and rebinding of the low molecular mass polypeptides to produce a functional Photosystem I complex.

AB - We had shown previously that addition of urea to a Synechococcus 6301 Photosystem I complex leads to dissociation of the 8.9 kDa, FA FB polypeptide, from the P-700- and FX-containing Photosystem I core protein (Golbeck et al. (1988) FEBS Lett. 240, 9-14). In the presence of chaotropes, the iron-sulfur clusters in the 8.9 kDa, FA FB polypeptide are unstable, and degrade to the level of zero-valence sulfur (Parrett et al. (1989) Biochim. Biophys. Acta 973, 324-332). We now report that addition of FeCl3, Na2S, and β-mercaptoethanol to a mixture of the low molecular mass polypeptides and the purified Photosystem I core protein results in complete restoration of light-induced charge separation between P-700 and FA FB, including (i) the 30 ms room temperature charge recombination between P-700+ and [ FA FB]- and (ii) the characteristic light-induced ESR spectrum of FA and FB with g values of 2.05, 1.94, 1.92 and 1.89. Analysis by SDS-PAGE shows that the reconstituted 8.9 kDa, FA FB polypeptide has rebound to the Photosystem I core protein. The purified Photosystem I core protein was treated with 3 M urea and 5 mM potassium ferricyanide to oxidatively denature FX to the level of zero-valence sulfur; light-induced charge separation in the apo-FX core protein results in a 3 μs optical transient due to the relaxation of the P-700 triplet state. Addition of FeCl3, Na2S and β-mercaptoethanol results in restoration of light-induced charge separation between P-700 and FX, including (i) the 1.2 ms room temperature charge recombination between P-700+ and FX - and (ii) the characteristic light-induced ESR resonances of FX with g values of 2.05, 1.86 and 1.78. Addition of FeCl3, Na2S and β-mercaptoethanol to a mixture of the FX and FA FB apoproteins results in reconstitution of electron flow from P-700 to FA FB, indicating quantitative reinsertion of the FX as well as the FA FB iron-sulfur clusters and quantitative rebinding of the 8.9 kDa polypeptide to the Photosystem I core protein. This reconstitution technique makes possible novel studies of Photosystem I, including chemical or genetic modification of the FX or FA FB apoproteins followed by reinsertion of the iron-sulfur clusters and rebinding of the low molecular mass polypeptides to produce a functional Photosystem I complex.

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