Modified ligands to FA and FB in photosystem I. I. Structural constraints for the formation of iron-sulfur clusters in free and rebound PsaC.
Cysteines 14, 21, 34, 51, or 58 in PsaC of photosystem I (PS I) were replaced with aspartic acid (C21D and C58D), serine (C14S, C34S, and C51S), and alanine (C14A, C34A, and C51A). When free in solution, the C34S and C34A holoproteins contained two S = 1/2 ground state [4Fe-4S] clusters; all other mutant proteins contained [3Fe-4S] clusters and [4Fe-4S] clusters; in addition, there was evidence in C14S, C51S, C14A, and C51A for high spin (S = 3/2) [4Fe-4S] clusters, presumably in the modified site. These findings are consistent with the assignment of C14, C21, C51, and C58, but not C34, as ligands to FA and FB. The [4Fe-4S] clusters in the unmodified sites in C14S, C51S, C14A, and C51A remained highly electronegative, with Em values ranging from -495 to -575 mV. The [3Fe-4S] clusters in the modified sites were driven 400 to 450 mV more oxidizing than the native [4Fe-4S] clusters, with Em values ranging from -98 mV to -171 mV. A C14D/C51D double mutant contains [3Fe-4S] and S = 1/2 [4Fe-4S] clusters, showing that the 3Cys.1Asp motif is also able to accommodate a low spin cubane. When C34S, C34A, C14S, C51S, C14A, and C51A were rebound to P700-FX cores, electron transfer to FA/FB was regained, but functional reconstitution has not yet been achieved for C21D, C58D, or C14D/C51D. These data imply that PsaC requires two iron-sulfur clusters to refold, one of which must be a cubane. Since two [4Fe-4S] clusters are found in all reconstituted PS I complexes, the presence of two cubanes in free PsaC may be a necessary precondition for binding to P700-FX cores.
D000595:Amino Acid Sequence; D002734:Chlorophyll / Q000378:metabolism; D000458:Cyanobacteria / Q000378:metabolism; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins; D008872:Microwaves; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010460:Peptococcus / Q000378:metabolism; D045331:Photosystem I Protein Complex; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D007506:Iron-Sulfur Proteins; D008024:Ligands; D008565:Membrane Proteins; D045331:Photosystem I Protein Complex; D011506:Proteins; D011994:Recombinant Proteins; C089909:photosystem I, psaB subunit; D002734:Chlorophyll; D003545:Cysteine