Site-Directed Conversion of a Cysteine to Aspartate Leads to the Assembly of a [3Fe-4S] Cluster in PsaC of Photosystem I. The Photoreduction of FA Independent of FB

Jindong Zhao, Donald Ashley Bryant, Ning Li, Patrick V. Warren, John H. Golbeck

Research output: Contribution to journalArticle

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Abstract

The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-Fx core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K. Similar to the behavior of the control complex at 15 K, only 12% of the Fb cluster is photoreduced in the complex reconstituted with C51D, whereas about 73% of the FA cluster is photoreduced in the complex reconstituted with C14D. On the basis of amino acid sequence similarities between PsaC and ferredoxins of known structure, we propose that the FA cluster is liganded by cysteines 21, 48, 51, and 54 while the FB cluster is liganded by cysteines 11, 14, 17, and 58. The ability to photoreduce FA in the presence of a nonfunctional FB indicates that the latter is not an obligatory intermediate in the pathway of electrons to FA.

Original languageEnglish (US)
Pages (from-to)5093-5099
Number of pages7
JournalBiochemistry
Volume31
Issue number22
DOIs
StatePublished - Feb 1 1992

Fingerprint

Photosystem I Protein Complex
Lighting
Aspartic Acid
Cysteine
Paramagnetic resonance
Mutant Proteins
Electrons
Ligands
Synechococcus
Dithionite
Ferredoxins
Mutagenesis
Temperature
Behavior Control
Aptitude
Site-Directed Mutagenesis
Sulfur
Escherichia coli
Amino Acid Sequence
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{9e10ec98c2454e1285d7859477ae5177,
title = "Site-Directed Conversion of a Cysteine to Aspartate Leads to the Assembly of a [3Fe-4S] Cluster in PsaC of Photosystem I. The Photoreduction of FA Independent of FB",
abstract = "The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-Fx core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K. Similar to the behavior of the control complex at 15 K, only 12{\%} of the Fb cluster is photoreduced in the complex reconstituted with C51D, whereas about 73{\%} of the FA cluster is photoreduced in the complex reconstituted with C14D. On the basis of amino acid sequence similarities between PsaC and ferredoxins of known structure, we propose that the FA cluster is liganded by cysteines 21, 48, 51, and 54 while the FB cluster is liganded by cysteines 11, 14, 17, and 58. The ability to photoreduce FA in the presence of a nonfunctional FB indicates that the latter is not an obligatory intermediate in the pathway of electrons to FA.",
author = "Jindong Zhao and Bryant, {Donald Ashley} and Ning Li and Warren, {Patrick V.} and Golbeck, {John H.}",
year = "1992",
month = "2",
day = "1",
doi = "10.1021/bi00137a001",
language = "English (US)",
volume = "31",
pages = "5093--5099",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
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}

Site-Directed Conversion of a Cysteine to Aspartate Leads to the Assembly of a [3Fe-4S] Cluster in PsaC of Photosystem I. The Photoreduction of FA Independent of FB . / Zhao, Jindong; Bryant, Donald Ashley; Li, Ning; Warren, Patrick V.; Golbeck, John H.

In: Biochemistry, Vol. 31, No. 22, 01.02.1992, p. 5093-5099.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Site-Directed Conversion of a Cysteine to Aspartate Leads to the Assembly of a [3Fe-4S] Cluster in PsaC of Photosystem I. The Photoreduction of FA Independent of FB

AU - Zhao, Jindong

AU - Bryant, Donald Ashley

AU - Li, Ning

AU - Warren, Patrick V.

AU - Golbeck, John H.

PY - 1992/2/1

Y1 - 1992/2/1

N2 - The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-Fx core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K. Similar to the behavior of the control complex at 15 K, only 12% of the Fb cluster is photoreduced in the complex reconstituted with C51D, whereas about 73% of the FA cluster is photoreduced in the complex reconstituted with C14D. On the basis of amino acid sequence similarities between PsaC and ferredoxins of known structure, we propose that the FA cluster is liganded by cysteines 21, 48, 51, and 54 while the FB cluster is liganded by cysteines 11, 14, 17, and 58. The ability to photoreduce FA in the presence of a nonfunctional FB indicates that the latter is not an obligatory intermediate in the pathway of electrons to FA.

AB - The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-Fx core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K. Similar to the behavior of the control complex at 15 K, only 12% of the Fb cluster is photoreduced in the complex reconstituted with C51D, whereas about 73% of the FA cluster is photoreduced in the complex reconstituted with C14D. On the basis of amino acid sequence similarities between PsaC and ferredoxins of known structure, we propose that the FA cluster is liganded by cysteines 21, 48, 51, and 54 while the FB cluster is liganded by cysteines 11, 14, 17, and 58. The ability to photoreduce FA in the presence of a nonfunctional FB indicates that the latter is not an obligatory intermediate in the pathway of electrons to FA.

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U2 - 10.1021/bi00137a001

DO - 10.1021/bi00137a001

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VL - 31

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JF - Biochemistry

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