The Photosystem I core protein containing P700, A₀, A₁, and Fx has been isolated from Synechococcus sp. PCC 6301 and spinach Photosystem I complexes with 6.8 M urea followed by sucrose density ultracentrifugtion. The Photosystem I core protein has retained > 90% of Fx and 100% of P700 (determined by optical spectroscopy) but is totally devoid of iron-sulfur centers Fᴀ and Fв (determined by optical and ESR spectroscopy). SDS-PAGE indicates the retention of the 82-and 83-kDa reaction center polypeptides. The loss of Fᴀ and Fв is further reflected in the decline of acid labile sulfide from 11.8 ± 0.4 S²-/P700 in the Photosystem I complex to 4.6 ± 0.3 S²-/P700 in the Photosystem I core.protein. This preparation represents the first isolation of an intact reaction center core incorporating the redox centers P700 through Fx but totally lacking Fᴀ and Fʙ. Complete restoration of electron flow from P700 to Fᴀ/Fв was achieved by incubating a P700 and Fx-containing Photosystem I core protein with a freshly isolated 8.9-kDa, Fᴀ/Fв polypeptide. When illuminated during freezing, both Fᴀ and Fв become quantitatively reduced, and the ESR spectrum is nearly indistinguishable from Fᴀ and Fв in the control Photosystem I complex. In the reconstituted Photosystem I complex Fx is photochemically reduced only in the presence of Fᴀ- and Fв- and the broad high field resonance of Fx in the core protein appears upon reconstitution to be indistinguishable from Fx in the control Photosystem I complex. Optical flash photolysis after extensive washing confirms the complete restoration of the P700+ [Fᴀ/Fв]- backreaction, indicating quantitative rebinding of the 8.9-kOa polypeptide. This procedure.represents the first reconstitution of the Photosystem I complex from a purified Photosystem I core protein and a freshly isolated 8.9 kDa Fᴀ/Fв holoprotein, and makes possible independent manipulation of the two subunits that carry the entire electron acceptor system of Photosystem I. The Fᴀ/Fв iron-sulfur clusters in the 8.9 kDa polypeptide are easily degraded to the level of zero-valence sulfur during isolation from the Photosystem I complex. The level of degradation is minimized by working under anaerobic conditions and low temperature. It has been found that incubation of the purified Photosystem I core protein and the low molecular mass polypeptides (which includes the 8.9-kDa Fᴀ/Fв apoprotein) with a solution of FeCI₃, Na₂S and B-mercaptoethanol restores the light induced charge separation between P700 and Fᴀ/Fв. The optical and spectroscopic properties are indistinguishable from the control Photosystem one I complex. When a rebuilt spinach or Synechococcus sp. PeC 6301 Fᴀ/Fв polypeptide is rebound to a Photosystem I core protein from the same species, the reconstituted complexes show light-induced ESR spectra of Fᴀ/Fв with g-values identical to their respective controls. However, when the rebuilt spinach Fᴀ/Fв polypeptide is rebound to a Synechococcus sp. PCC 6301 Photosystem I core protein, the hybrid spinach Synechococcus sp. PCC 6301 Photo system complex shows a light-induced ESR spectrum of Fᴀ/Fв with g- values that differ slightly, but characteristically, from those of both spinach and Synechococcus sp. PCC 6301 control complexes. The hybrid spinach-Synechococcus sp. PCC 6301 Photosystem I complex was completely functional in light-induced charge separation between P700 and Fᴀ/Fв and showed a normal 30-ms room temperature charge recombination between P700+ and [Fᴀ/Fв]-. Accordingly, rebuilding of the Fᴀ and Fв iron-sulfur clusters is now possible and reconstitution of the Fᴀ/Fв holoprotein after chemical modification of the Fᴀ/Fв apoprotein or genetic modification of the psaC gene should reveal new findings about Photosystem I structure and function.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-2255 |
| Date | 01 January 1990 |
| Creators | Mehari, Tetemke |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
Page generated in 0.0362 seconds