Photosystem I (PS I) mediates electron-transfer from plastocyanin to ferredoxin via a photochemically active chlorophyll dimer (P700), a monomeric chlorophyll electron acceptor (A0), a phylloquinone (A1), and three [4Fe-4S] clusters (F(X/A/B)). The sequence of electron-transfer events between the iron-sulfur cluster, F(X), and ferredoxin is presently unclear. Owing to the presence of a 2-fold symmetry in the PsaC protein to which the iron-sulfur clusters F(A) and F(B) are bound, the spatial arrangement of these cofactors with respect to the C2-axis of symmetry in PSI is uncertain as well. An unequivocal determination of the spatial arrangement of the iron- sulfur clusters F(A) and F(B) within the protein is necessary to unravel the complete electron-transport chain in PS I. In the present study, we generate EPR signals from charge-separated spin pairs (P700/+-F(X/A/B)/(red)) in PSI and characterize them by progressive microwave power saturation measurements to determine the arrangement of the iron-sulfur clusters F(X/A/B)relative to P700. The microwave power at half saturation (P(1/2)) of P700/+ is greater when both F(A) and F(B) are reduced in untreated PSI than when only F(A) is reduced in mercury-treated PS I. The experimental P(1/2) values are compared to values calculated by using P700-F(A/B) crystallographic distances and assuming that either F(A) or F(B) is closer to P700/+. On the basis of this comparison of experimental and theoretical values of spin relaxation enhancement effects on P700/+ in P700/+ [4Fe-4S]- charge-separated pairs, we find that iron-sulfur cluster F(A) is in closer proximity to P700 than the F(B) cluster.
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