Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors FA and FB in Heliobacterium modesticaldum

Mark Heinnickel, Gaozhong Shen, John H. Golbeck

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24 Citations (Scopus)

Abstract

The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates F A- and FB-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichla coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-FX cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [FA/F B]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798 + and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.

Original languageEnglish (US)
Pages (from-to)2530-2536
Number of pages7
JournalBiochemistry
Volume46
Issue number9
DOIs
StatePublished - Mar 6 2007

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Photosystem I Protein Complex
Ferredoxins
Ports and harbors
Paramagnetic resonance
Genes
Electrons
Iron-Sulfur Proteins
Photosynthetic Reaction Center Complex Proteins
Amino Acids
Apoproteins
Gene encoding
Cloning
Amino Acid Sequence
Sulfur
Bacteria
Iron
Anaerobic Bacteria
Lighting
Gels
Gram-Positive Bacteria

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors FA and FB in Heliobacterium modesticaldum",
abstract = "The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates F A- and FB-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichla coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-FX cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [FA/F B]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798 + and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.",
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T1 - Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors FA and FB in Heliobacterium modesticaldum

AU - Heinnickel, Mark

AU - Shen, Gaozhong

AU - Golbeck, John H.

PY - 2007/3/6

Y1 - 2007/3/6

N2 - The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates F A- and FB-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichla coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-FX cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [FA/F B]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798 + and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.

AB - The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates F A- and FB-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichla coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-FX cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [FA/F B]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798 + and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.

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