Reconstitution of the iron-sulfur clusters in the isolated F A/F B protein: EPR spectral characterization of same-species and cross-species Photosystem I complexes

Abstract

The ESR spectra of the F A /F B iron-sulfur clusters in spinach and Synechococcus sp. PCC 6301 Photosystem I complexes differ slightly, but characteristically. In the fully reduced state, the spinach F A /F B resonances appear at g = 2.051, 1.941, 1.923, 1.887, while the Synechococcus sp. PCC 6301 F A /F B 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 F A /F B 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 F A /F B apoprotein for 12 h with FeCl 3 and Na 2 S in the presence of β-mercaptoethanol under strictly anaerobic conditions. Upon chemical reduction with Na 2 S 2 O 4 at pH 10, the rebuilt spinach F A /F B protein shows the broadened resonances of F A and F B , 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 F A /F B holoprotein is rebound to a Photosystem I core protein from the same species, the F A /F B 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 F A /F B 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 F A /F B holoprotein incurred during isolation. This in vitro reconstitution protocol circumvents the need to isolate the labile F A /F B holoprotein, and makes possible reinsertion of the iron-sulfur clusters following modification of the F A /F B apoprotein or the psaC gene.