The ESR spectra of the FA/FB iron-sulfur clusters in spinach and Synechococcus sp. PCC 6301 Photosystem I complexes differ slightly, but characteristically. In the fully reduced state, the spinach FA/FB resonances appear at g = 2.051, 1.941, 1.923, 1.887, while the Synechococcus sp. PCC 6301 FA/FB resonances appear at g = 2.047, 1.937, 1.917 and 1.879; both are independent of the details of Photosystem I isolation or the method of reduction. When the spinach or Synechococcus sp. PCC 6301 FA/FB holoprotein is removed from the thylakoid membrane by solvent extraction, the ESR resonances broaden and the iron-sulfur clusters become labile, degrading rapidly to the level of zero-valence sulfur. We show that the clusters can be reinserted in vitro by incubating the isolated FA/FB apoprotein for 12 h with FeCl3 and Na2S in the presence of β-mercaptoethanol under strictly anaerobic conditions. Upon chemical reduction with Na2S2O4 at pH 10, the rebuilt spinach FA/FB protein shows the broadened resonances of FA and FB, but when rebound to a Synechococcus sp. PCC 6301 Photosystem I core protein, the hybrid spinach-Synechococcus Photosystem I complex shows sharpened resonances with g-values of 2.052, 1.941, 1.922 and 1.886. These g-values are similar to those of the native spinach Photosystem I complex. In contrast, when a spinach or Synechococcus sp. PCC 6301 FA/FB holoprotein is rebound to a Photosystem I core protein from the same species, the FA/FB resonances appear identical to their respective control Photosystem I complexes. These results indicate that the altered ESR spectrum reported earlier on reconstitution of a solvent-extracted spinach FA/FB holoprotein with a Synechococcus sp. PCC 6301 Photosystem I core protein (Golbeck et al. (1988) FEBS Lett. 240, 9-14) is a consequence of the cross-species reconstitution and does not result from damage to the FA/FB holoprotein incurred during isolation. This in vitro reconstitution protocol circumvents the need to isolate the labile FA/FB holoprotein, and makes possible reinsertion of the iron-sulfur clusters following modification of the FA/FB apoprotein or the psaC gene.
All Science Journal Classification (ASJC) codes
- Cell Biology