The photoreduction of the oxidized and the semiquinone form of flavodoxin by photosystem I particles (PSI) from the wild type and a psaE deletion strain from the cyanobacterium Synechocystis sp. PCC 6803 was analyzed by flash-absorption spectroscopy to investigate a possible involvement of the PsaE subunit in this photoreduction process. The kinetics of the reduction of oxidized flavodoxin display a single-exponential component for both PSI preparations. Limiting electron transfer rates kobs of approximately 500 and approximately 900 s -1 are deduced for the wild type and PSI from the psaE-less mutant, respectively, indicating that the PsaE subunit is not important for this photoreduction process. In the case of wild-type PSI, the reduction of flavodoxin semiquinone is a biphasic process, displaying a fast first-order phase with a t1/2 of approximately 13 micro(s) which is then followed by a slower, concentration-dependent phase, for which a second-order rate constant k2 of 2.2 x 10(8) M-1 cm-1 is calculated. In contrast, photoreduction of the semiquinone by PSI from the psaE-less mutant is monoexponential, displaying only one second-order component with a second-order rate constant similar to those observed for wild-type PSI (k2 = 1.5 x 10(8) M-1 cm-1). The fast first-order component which is interpreted as an electron transfer process within a preformed complex between flavodoxin semiquinone and PSI is almost completely absent in the reduction of flavodoxin by the PsaE-less PSI. A similar loss of the fast phase is also observed for the photoreduction of flavodoxin semiquinone by PSI from a Synechococcus elongatus psaE-less mutant. Upon reconstitution of isolated PsaE to the PsaE-less PSI in vitro, approximately 80% of the fast first-order kinetic component is recovered, indicating that PsaE is required for high-affinity binding of the flavodoxin semiquinone to PSI. In addition, chemical cross-linking assays show that flavodoxin can no longer be cross-linked to PSI in detectable amounts when PsaE is missing on the reaction center. Taken together, these experiments indicate that the PsaE subunit is required for complex formation between PSI and flavodoxin but is not required for an efficient forward electron transfer from photosystem I to both forms of flavodoxin.
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