PsaC-C14D (cysteine 14 replaced by aspartic acid) contains a [3Fe-4S] and a [4Fe-4S] cluster in the FB and FA sites of the free protein [Yu, L., Zhao, J., Lu, W., Bryant, D. A., & Golbeck, J. H. (1993) Biochemistry 32, 8251-8258]. When PsaC-C14D is rebound to a photosystem I (PS I) core, the g-values of 2.043, 1.939, and 1.853 appear similar to FA in a wild-type PS I complex [Zhao, J. D., Li, N., Warren, P. V., Golbeck, J. H., & Bryant, D. A. (1992) Biochemistry 31, 5093-5099], The reconstituted PsaCC14D- PS I complex does not contain a [3Fe-4S] cluster; rather, a set of resonances with a rhombic line shape, a gm of ~1.97, and broad line widths indicate the presence of a mixed-ligand [4Fe-4S] cluster, termed FB‘, in the aspartate site. Both FA and FB‘ become photoreduced at 15 K, and show an interaction spectrum when reduced within the same reaction center. An electrochemical redox study shows that FA and FB‘ titrate with midpoint potentials near -600 mV at pH 10.0. Single-turnover flash experiments indicate that FA and FB‘ function as efficient electron acceptors at room temperature, and NADP+ photoreduction rates are about 70% that of a reconstituted PsaC-PS I complex. A population of 5 = 3/2, [4Fe-4S] clusters was tentatively identified in the free PsaC-C14D protein by characteristic EPR resonances in the g = 5.3 region. It is proposed that P700-FX cores have a high affinity for PsaC-C14D that contains two cubane clusters, and that on rebinding, the mixed-ligand cluster in the FB‘ site crosses over from the S = 3/2 to the S = 1/2 spin state. These results show that electron throughput from FX to ferredoxin in the PsaC-C14D-PS I complex remains efficient even though the spectroscopic and thermodynamic properties of FA and FB‘ are altered by the presence of the mixed-ligand iron-sulfur cluster in the FB site.
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