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., & McInt osh, L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,1132-1136] that the si te-directed conversion of cysteine-565 to serine (C565S) in PsaB of Sy nechocystis sp. PCC 6803 leads to an accumulation of photosystem I pol ypeptides and the low-temperature photoreduction of the terminal elect ron acceptors F(A) and F(B). In this paper, we report the occurrence o f a [3Fe-4S]1+,0 cluster in dodecyl maltoside-solubilized photosystem I complexes prepared from the C565S mutant. The [3Fe-4S] cluster is re ducible with dithionite at pH 6.5, implying a midpoint potential consi derably more oxidizing than either F(A) or F(B). Similar to the behavi or of F(X), the [3Fe-4S] cluster undergoes partial, reversible photore duction when the complex is illuminated at 15 K, and complete photored uction 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 photoreducti on of F(A) and F(B) in the C565S complex. Although the F(A) and F(B) r esonances are more intense when the complex is frozen during illuminat ion, they still account for <60% of F(A) and F(B) found by chemical re duction. When the F(A) and F(B) clusters are prereduced with dithionit e 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 spe cies 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 p otential low enough to transfer an electron to F(A) and F(B). We sugge st that F(X)' is assembled as a functional [4Fe-4S] cluster in vivo bu t in a minority of centers F(X)' loses an iron, resulting in a populat ion 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 tr ansfer from A1- to F(A) and F(B).