Abstract

The electron spin polarization (ESP) EPR signal arising from the P-700 + A 1 − radical pair in Photosystem I (PS I) consisting of the oxidized PS I primary donor, P-700 , and the reduced vitamin K 1 (K 1) acceptor, A 1, is studied as a function of isotopic labelling of the native A 1 acceptor. No pre-extraction of the native A 1 acceptor is utilized, instead, exchange of the native acceptor with protonated or perdeuterated K 1 is accomplished by incubation of the reaction centers in solution (incubation/exchange). The incubation/exchange process is studied in PS I of spinach and in two cyanobacteria, the thermophilic Synechococcus lividus ( S. lividus) and the non-thermophilic Synechococcus leopoliensis ( S. leopoliensis). A complete data set, HP HA 1, HP DA 1, DP HA 1, DP DA 1 (where P refers to the primary chlorophyll donor, A 1, the quinone acceptor, and H and D denote protonated and deuterated, respectively), is obtained for S. lividus. The correlated radical pair polarization model (CRPP) is used to reproduce the experimental lineshapes for the four different isotopic combinations of the P-700 +A 1 − radical pair. The experimental ESP EPR spectra are well predicted using the geometric and energetic parameters which have been deduced for perdeuterated PS I reaction centers in whole cells of S. lividus (Kothe, G. et al. (1994) J. Phys. Chem. 98, 2706–2712). The results suggest that the isotopically labelled K 1 binds in the A 1 site with the same orientation as the native acceptor in unexchanged PS I. No evidence for electron transfer to K 1 in configurations other than the A 1 binding site is observed. Incubation/exchange is studied as a function of incubation temperature. For spinach and non-thermophilic S. leopoliensis the effect of incubation/exchange is most efficient for 1/2 h incubations at temperatures around and above 30–35°C, but requires somewhat higher incubation temperatures, above 40–45°C, for thermophilic S. lividus. Similar changes in the ESP spectrum are also demonstrated for PS I reaction centers in thylakoid membranes of S. lividus.

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