Modified ligands to F_A and F_B in photosystem I: II. Characterization of a mixed ligand [4Fe-4S] cluster in the C51D mutant of PsaC upon rebinding to P700-F_X cores

A Photosystem I (PS I) complex reconstituted with PsaC-C51D (aspartate in lieu of cysteine in position 51) shows light-induced EPR signals with g values, line widths, and photoreduction behavior characteristic of F_B. Contrary to an earlier report, a [3Fe-4S] cluster was not located in the reconstituted PS I complex. Instead, a second set of resonances with g values of 2.044, 1.942, and 1.853 becomes EPR-visible when the C51D-PS I complex is measured at 4.2 K. This fast relaxing center, termed F_A′ is likely to represent a [4Fe-4S] cluster in the mixed ligand (3Cys·1Asp) site. Redox studies show that the E_m of F_A′ and F_B are -630 mV and -575 mV, respectively. Room temperature optical studies support the presence of two functioning electron acceptors subsequent to F_x, and NADP⁺ photoreduction rates mediated by ferredoxin and flavodoxin are nearly equivalent to the wild type. In addition to [3Fe-4S] clusters and S = 1/2 ground state [4Fe-4S] clusters, the free PsaC-C51D protein shows resonances near g = 5.5, which may represent a population of high spin (S = 3/2) [4Fe-4S] clusters in the mixed ligand F_A′ site. Similar to the C14D-PS I mutant complex, it is proposed that the P700-F_X core selectively rebinds those free PsaC-C51D proteins that contain two [4Fe-4S] clusters. These studies show that primary photochemistry and electron transfer rates in PS I are relatively unaffected by the presence of a highly reducing, mixed ligand cluster in the F_A′ site.