ABSTRACT
Photosystem I (PS I) converts the energy of light into chemical energy via transmembrane charge separation. The terminal electron transfer cofactors in PS I are three low-potential [4Fe-4S] clusters named F(X), F(A) and F(B), the last two are bound by the PsaC subunit. We have modelled the F(A) and F(B) binding sites by preparing two apo-peptides (maquettes), sixteen amino acids each. These model peptides incorporate the consensus [4Fe-4S] binding motif along with amino acids from the immediate environment of the iron-sulfur clusters F(A) and F(B). The [4Fe-4S] clusters were successfully incorporated into these model peptides, as shown by optical absorbance, EPR and Mössbauer spectroscopies. The oxidation-reduction potential of the iron-sulfur cluster in the F(A)-maquette is -0.44+/-0.03 V and in the F(B)-maquette is -0.47+/-0.03 V. Both are close to that of F(A) and F(B) in PS I and are considerably more negative than that observed for other [4Fe-4S] model systems described earlier (Gibney, B. R., Mulholland, S. E., Rabanal, F., and Dutton, P. L. Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 15041-15046). Our optical data show that both maquettes can irreversibly bind to PS I complexes, where PsaC-bound F(A) and F(B) were removed, and possibly participate in the light-induced electron transfer reaction in PS I.
Subject(s)
Iron-Sulfur Proteins/chemistry , Peptides/chemistry , Photosystem I Protein Complex/chemistry , Amino Acid Sequence , Apoproteins/chemistry , Apoproteins/metabolism , Binding Sites , Chlorophyll/chemistry , Chlorophyll/metabolism , Electron Spin Resonance Spectroscopy , Iron-Sulfur Proteins/metabolism , Kinetics , Models, Molecular , Molecular Sequence Data , Oxidation-Reduction , Peptides/chemical synthesis , Peptides/metabolism , Photosystem I Protein Complex/metabolism , Protein Binding , Spectroscopy, MossbauerABSTRACT
Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S](1+,2+) cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S](1+,2+) clusters termed F(A) and F(B). In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F(B). Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S](1+,2+) clusters, despite the absence of one of the biological ligands. (19)F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F(B) cluster. The finding that site-modified [4Fe-4S](1+,2+) clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.
