EVIDENCE FOR A MIXED-LIGAND [4FE-4S] CLUSTER IN THE C14D MUTANT OF PSAC - ALTERED REDUCTION POTENTIALS AND EPR SPECTRAL PROPERTIES OF THE F-A AND F-B CLUSTERS ON REBINDING TO THE P700-F-X CORE

PsaC-C14D (cysteine 14 replaced by aspartic acid) contains a [3Fe-4S] and a [4Fe-4S] cluster in the F-B and F-A sites of the free protein [Yu, L., Zhao, J., Lu, W., Bryant, D. A., and 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 F-A in a wild-type PS I complex [Zhao, J. D., Li, N., Warren, P. V., Golbeck, J. H., and Bryant, D. A. (1992) Biochemistry 31, 5093-5099]. The reconstituted PsaC-C14D-PS I complex does not contain a [3Fe-4S] cluster; rather, a set of resonances with a rhombic line shape, a g(av) of similar to 1.97, and broad line widths indicate the presence of a mixed-ligand [4Fe-4S] cluster, termed F-B', in the aspartate site. Both F-A and F-B' become photoreduced at 15 K, and show an interaction spectrum when reduced within the same reaction center. An electrochemical redox study shows that F-A and F-B' titrate with midpoint potentials near -600 mV at pH 10.0. Single-turnover flash experiments indicate that F-A and F-B' function as efficient electron accepters at room temperature, and NADP(+) photoreduction rates are about 70% that of a reconstituted PsaC-PS I complex. A population of S = 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-F-X cores have a high affinity for PsaC-C14D that contains two cubane clusters, and that on rebinding, the mixed-ligand cluster in the F-B' Site crosses over from the S = 3/2 to the S = 1/2 spin state. These results show that electron throughput from F-X to ferredoxin in the PsaC-C14D-PS I complex remains efficient even though the spectroscopic and thermodynamic properties of F-A and F-B' are altered by the presence of the mixed-ligand iron-sulfur cluster in the F-B site.