In a previous paper we showed that the C51D mutant of PsaC contains a [3Fe-4S] cluster in the FA site and a [4Fe-4S] cluster in the FB site and that the C14D mutant contains an uncharacterized cluster in the FB site and a [4Fe-4S] cluster in the FA site [Zhao, J. D., Li, N., Warren, P. V., Golbeck, J. H., & Bryant, D. A. (1992) Biochemistry 31, 5093–5099]. In this paper we describe the electrochemical and electron spin resonance properties of the recombinant C14D and C51D holoproteins after in vitro reinsertion of the iron-sulfur clusters. Unbound PsaC shows no significant resonances in the oxidized state, but the unbound C14D and C51D mutant proteins show an intense set of resonances at g ∼ 2.02 and 1.99 characteristic of an oxidized [3Fe-4S]1+/0 cluster. The Em′ values for the [3Fe-4S]1+/0 clusters in C14D (Fb*) and C51D (FA*) are −98 mV, and both represent one-electron transfers. After reduction with dithionite at pH 10.0, wild-type PsaC shows a broad set of resonances resulting from the superposition of FA− and FB− characterized by a low-field peak at an apparent g value of 2.051 and a high-field trough at an apparent g value of 1.898. The FB resonances in C51D were slightly narrower, with a low-field peak at an apparent g value of 2.039 and high-field trough at an apparent g value of 1.908. The FA resonances in C14D were somewhat broader, with a low-field peak at an apparent g value of 2.042 and a high-field trough at an apparent g value of 1.898. The Em′ value for FA in C14D is −515 mV, and the Em′ value for FB in C51D is −580 mV; both represent one-electron transfers. These values are nearly identical to the Em′ values determined for the FA and FB clusters in PsaC bound to the photosystem I complex. This result indicates that the midpoint potentials of FA and FB are determined solely by the primary amino acid sequence of PsaC and not by interaction with PsaD or with the photosystem I core.
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