SITE-DIRECTED CONVERSION OF CYSTEINE-565 TO SERINE IN PSAB OF PHOTOSYSTEM-I RESULTS IN THE ASSEMBLY OF [3FE-4S] AND [4FE-4S] CLUSTERS IN F(X) - A MIXED-LIGAND [4FE-4S] CLUSTER IS CAPABLE OF ELECTRON-TRANSFER TO F(A) AND F(B)

We reported earlier [Smart, L.B., Warren, P.V., Golbeck, J.H., & McIntosh, L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,1132-1136] that the site-directed conversion of cysteine-565 to serine (C565S) in PsaB of Synechocystis sp. PCC 6803 leads to an accumulation of photosystem I polypeptides and the low-temperature photoreduction of the terminal electron acceptors F(A) and F(B). In this paper, we report the occurrence of a [3Fe-4S]1+,0 cluster in dodecyl maltoside-solubilized photosystem I complexes prepared from the C565S mutant. The [3Fe-4S] cluster is reducible with dithionite at pH 6.5, implying a midpoint potential considerably more oxidizing than either F(A) or F(B). Similar to the behavior of F(X), the [3Fe-4S] cluster undergoes partial, reversible photoreduction when the complex is illuminated at 15 K, and complete photoreduction when the sample is illuminated during freezing. Contrary to the result expected in the presence of a relatively high-potential F(X), there is significant low-temperature and room temperature photoreduction of F(A) and F(B) in the C565S complex. Although the F(A) and F(B) resonances are more intense when the complex is frozen during illumination, they still account for <60% of F(A) and F(B) found by chemical reduction. When the F(A) and F(B) clusters are prereduced with dithionite at pH 10.0, a new set of resonances appear upon illumination at g = 2.015, 1.941, and 1.811, and disappear on subsequent darkness. The species giving rise to this signal is most likely a mixed-ligand [4Fe-4S]2+,1+ Cluster located in the F(X) site. This acceptor, denoted F(X)', has different ESR properties from wild-type F(X), but it has a redox potential low enough to transfer an electron to F(A) and F(B). We suggest that F(X)' is assembled as a functional [4Fe-4S] cluster in vivo but in a minority of centers F(X)' loses an iron, resulting in a population of [3Fe-4S] clusters which cannot pass the electron forward to F(A) or F(B). The redox behavior of the [3Fe-4S] and mixed-ligand [4Fe-4S] clusters is consistent with the participation of F(X) in electron transfer from A1- to F(A) and F(B).