Purification of the photosynthetic reaction center from <Emphasis Type="Italic">Heliobacterium modesticaldum</Emphasis> |
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Authors: | Iosifina Sarrou Zahid Khan John Cowgill Su Lin Daniel Brune Steven Romberger John H Golbeck Kevin E Redding |
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Institution: | (1) Department of Chemistry and Biochemistry, Arizona State University, 1711 S. Rural Rd., Tempe, AZ 85287-1604, USA;(2) Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA;(3) School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA;(4) Department of Biochemistry and Molecular Biology, and Department of Chemistry, The Pennsylvania State University, University Park, PA 16801, USA; |
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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 c
553 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 a
F, 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 K
M of 10 μM and a k
cat 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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