Purification of the photosynthetic reaction center from Heliobacterium modesticaldum.

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

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)291-302
Number of pages12
JournalPhotosynthesis Research
Volume111
Issue number3
DOIs
StatePublished - 2012

Fingerprint

Heliobacterium
photosynthetic reaction centers
Photosynthetic Reaction Center Complex Proteins
Purification
polypeptides
Bacteriochlorophylls
Peptides
Light
ABC transporters
Flavodoxin
cytochrome c
menaquinones
Dithionite
Membranes
Vitamin K 2
anaerobic conditions
purity
biochemistry
Biochemistry
ATP-Binding Cassette Transporters

ASJC Scopus subject areas

  • Biochemistry
  • Plant Science
  • Cell Biology

Cite this

Purification of the photosynthetic reaction center from Heliobacterium modesticaldum. / Sarrou, Iosifina; Khan, Zahid; Cowgill, John; Lin, Su; Brune, Daniel; Romberger, Steven; Golbeck, John H.; Redding, Kevin.

In: Photosynthesis Research, Vol. 111, No. 3, 2012, p. 291-302.

Research output: Contribution to journalArticle

Sarrou, Iosifina ; Khan, Zahid ; Cowgill, John ; Lin, Su ; Brune, Daniel ; Romberger, Steven ; Golbeck, John H. ; Redding, Kevin. / Purification of the photosynthetic reaction center from Heliobacterium modesticaldum. In: Photosynthesis Research. 2012 ; Vol. 111, No. 3. pp. 291-302.
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