Purification of the photosynthetic reaction center from Heliobacterium modesticaldum View Full Text


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Article Info

DATE

2012-03-02

AUTHORS

Iosifina Sarrou, Zahid Khan, John Cowgill, Su Lin, Daniel Brune, Steven Romberger, John H. Golbeck, Kevin E. Redding

ABSTRACT

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c553 and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 81-OH-Chl aF, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the FA and FB [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P800+FX− state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a KM of 10 μM and a kcat of 9.5 s−1 under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light. More... »

PAGES

291-302

References to SciGraph publications

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  • 2006-05-12. Tricine–SDS-PAGE in NATURE PROTOCOLS
  • 2010-05-26. Identification and characterization of PshBII, a second FA/FB-containing polypeptide in the photosynthetic reaction center of Heliobacterium modesticaldum in PHOTOSYNTHESIS RESEARCH
  • 2011-04-17. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å in NATURE
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  • 2007-04-25. Heliobacterial photosynthesis in PHOTOSYNTHESIS RESEARCH
  • 2012-02-02. The FX iron–sulfur cluster serves as the terminal bound electron acceptor in heliobacterial reaction centers in PHOTOSYNTHESIS RESEARCH
  • 2001-06. Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution in NATURE
  • 1995-04. Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils in ARCHIVES OF MICROBIOLOGY
  • 2003-12. Crystal structure of plant photosystem I in NATURE
  • Identifiers

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    http://scigraph.springernature.com/pub.10.1007/s11120-012-9726-9

    DOI

    http://dx.doi.org/10.1007/s11120-012-9726-9

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    https://www.ncbi.nlm.nih.gov/pubmed/22383054


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    30 schema:description We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c553 and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 81-OH-Chl aF, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the FA and FB [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P800+FX− state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a KM of 10 μM and a kcat of 9.5 s−1 under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.
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