title: Purification of the photosynthetic reaction center from Heliobacterium modesticaldum.
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 8(1)-OH-Chl a (F), and one 4,4'-diaponeurosporene. It lacks the PshB polypeptide binding the F(A) and F(B) [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 P(800) (+)F(X)(-) 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.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University, 1711 S. Rural Rd., Tempe, AZ 85287-1604, USA.
authors.forename: Iosifina
authors.initials: I
authors.lastname: Sarrou
authors.forename: Zahid
authors.initials: Z
authors.lastname: Khan
authors.forename: John
authors.initials: J
authors.lastname: Cowgill
authors.forename: Su
authors.initials: S
authors.lastname: Lin
authors.forename: Daniel
authors.initials: D
authors.lastname: Brune
authors.forename: Steven
authors.initials: S
authors.lastname: Romberger
authors.forename: John H
authors.initials: JH
authors.lastname: Golbeck
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Redding
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification; D001429:Bacteriochlorophylls / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000737:chemistry* / Q000378:metabolism; D008027:Light; D010100:Oxygen; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D001429:Bacteriochlorophylls; D002352:Carrier Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D010100:Oxygen