The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(x) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(x), we used single-turnover flashes to promote step-by-step electron transfer between electron carders in control (both F(A) and F(B) present) and HgCl2- treated (F(B)-less) PS I complexes from Synechococcus sp: PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferrtedoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)/- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin, in the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)/-, in the presence of a slow donor, 2,6-dichtorophenol-indophenol. This recombination remained the only pathway of F(A)/-, reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)/- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild- type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(x) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(x) and F(B) as the distal cluster that donates electrons to ferredoxin.
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