Additive Effect of Mutations Affecting the Rate of Phylloquinone Reoxidation and Directionality of Electron Transfer within Photosystem I † Stefano Santabarbara 1,2,3‡ , Audrius Jasaitis 1,2,‡§ , Martin Byrdin 2§ , Feifei Gu 1– , Fabrice Rappaport 2 and Kevin Redding* 1,2,3 1 Department of Chemistry, University of Alabama, Tuscaloosa, AL 2 Institut de Biologie Physico-Chimique, UMR7141 CNRS-Univ. Paris 6, Paris, France 3 Department of Chemistry & Biochemistry, Arizona State University, Tempe, AZ Received 9 May 2008, accepted 25 August 2008, DOI: 10.1111 ⁄ j.1751-1097.2008.00458.x ABSTRACT Optical pump-probe spectroscopy in the nanosecond–micro- second timescale has been used to study the electron transfer reactions taking place within the Photosystem I reaction center of intact Chlamydomonas reinhardtii cells. The biphasic kinetics of phylloquinone (PhQ) reoxidation were investigated in double mutants that combine a mutation (PsaA-Y696F) near the primary acceptor chlorophyll, ec3 A , with those near PhQ A (PsaA-S692A, PsaA-W697F). The PsaA-S692A and PsaA-W697F mutations selectively lengthened the 200 ns lifetime component observed in the wild-type (WT). The 320 ns component was unaltered in the single mutant, both in terms of lifetime and relative amplitude. However, both double mutants possessed a 320 ns component (PhQ B - reoxidation) with increased amplitude compared with the WT and the individual PhQ A mutants. The component assigned to PhQ A - reoxidation was slowed, like the individual PhQ A mutants, and of lower amplitude, as observed in the single ec3 A mutant. Hence, the effects of these mutations are almost entirely additive, providing strong support for the previously proposed bidirectional electron transfer model, which attributes the 320 and 3200 ns phases to reoxidation of PhQ B or PhQ A , respec- tively. Moreover, in all the mutants investigated, it was also possible to observe an intermediate (180 ns) component, as previously reported for mutants of the PhQ A binding pocket (Biochim. Biophys. Acta [2006] 1757, 1529–1538), which we have tentatively attributed to forward electron transfer between the iron–sulfur clusters F X and F A ⁄ B . INTRODUCTION Photosystem I (PS1) is a large macromolecular complex indispensable for oxygenic photosynthesis. In higher plants it is located in the thylakoid membranes of the chloroplast. The overall catalytic activity of PS1 is the light-driven oxidation of plastocyanin and the reduction of ferredoxin, which are both soluble electron carriers. The cofactors involved in primary charge separation events are all chlorophyll a molecules (for recent data on primary charge separation see refs. [1–3]), while the terminal acceptors are [4Fe-4S] clusters (4,5). Phylloqui- none (PhQ, also called ‘‘A 1 ’’) acts as an intermediate electron acceptor. It is reduced by the cofactor located upstream in the electron transfer chain (ec3 or ‘‘A 0 ’’) in tens of picoseconds (6–9). The phyllosemiquinone radical (PhQ ) ) displays complex and polyphasic reoxidation kinetics in the nanosecond time scale (reviewed in refs. [10–12]). It was shown that, in isolated PS1 particles, the reoxidation kinetics of the semiphylloqui- none radical are described by at least two characteristic lifetimes of 20 and 200 ns. Although biphasic kinetics of PhQ ) reoxidation were observed in vitro with purified PS1 particles from plants (13) and cyanobacteria (14–16), they were largely attributed to structural heterogeneity at the level of PhQ ⁄ F X , as the amplitude of the faster phase exhibited large intersample variability and was shown to increase as a result of treatment with strong detergents (17). Alternatively, the biphasic decay of PhQ ) was interpreted in terms of a small equilibrium constant for the reaction involving this species and the successive electron acceptor, the iron–sulfur cluster F X (10,14). Using a new spectrometer, Joliot and Joliot (18) were able to observe biphasic PhQ ) reoxidation kinetics within PS1 in intact green algal cells, ruling out the possibility that the 20 ns reoxidation phase arose from perturbation of the samples during isolation of the complex. Based on thermo- dynamic considerations on the effect of electric field gradi- ents on the driving force for phyllosemiquinone reoxidation, these authors argued against the suggestion of a small driving force for PhQ ) reoxidation (18). It was instead proposed that the phylloquinones bound by either the PsaA (PhQ A ) or PsaB (PhQ B ) subunits were both active in electron transfer. This hypothesis, which is often referred to as the bidirectional electron transfer model, received substantial experimental support by the observation that mutations near PhQ A in Chlamydomonas reinhardtii specifically slowed the kinetics of the slower (200 ns) reoxidation phase, while mutations near PhQ B affected only the faster (20 ns) †This invited paper is part of the Symposium-in-Print: Photosynthesis. ‡These authors contributed equally to this work. §Current addresses: Audrius Jasaitis, 7 Domaine de la Boutte a la Reine, 91120 Palaiseau, France. Martin Byrdin: DBCM ⁄ SBE, Baˆtiment 532, Pie`ce, CEA Saclay, 91191 Gif-sur- Yvette, France. –Died on 18 July 2007, of a brain aneurism, after having collapsed in her postdoctoral laboratory at the University of Alabama (Birmingham). This paper is dedicated to her memory. *Corresponding author email: kevin.redding@asu.edu (Kevin Redding) Ó 2008 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/08 Photochemistry and Photobiology, 2008, 84: 1381–1387 1381