PsaC subunit of photosystem I is oriented with iron-sulfur cluster FB as the immediate electron donor to ferredoxin and flavodoxin

The PsaC subunit of photosystem t (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 carriers in control (both F-A and F-B present) and HgCl2-treated (F-B-less) PS I complexes from Synechococcus so. 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 ferredoxin, 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-dichlorophenol-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.