Biochemical and Biophysical Characterization of Photosystem I from Phytoene Desaturase and ζ-Carotene Desaturase Deletion Mutants of Synechocystis Sp. PCC 6803

Abstract

In photosystem I, oxidation of reduced acceptor A1 ⴚ through iron-sulfur cluster FX is biphasic with half- times of⬃5–30 ns (“fast” phase) and ⬃150–300 ns (“slow” phase). Whether these biphasic kinetics reflect unidirec- tional electron transfer, involving only the PsaA-side phylloquinone or bi-directional electron transfer, in- volving both the PsaA- and PsaB-side phylloquinones, has been the source of some controversy. Brettel (Brettel, K. (1988) FEBS Lett. 239, 93–98) and Joliot and Joliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130–11136) have attributed to nearby carotenoids electrochromic band shifts, accompanying A1 reduction, centered at ⬃450 and 500–510 nm. As a test of these assignments, we separately deleted in Synechocystis sp. PCC 6803 the genes that encode phytoene desaturase (encoded by crtP (pds)) and ?-carotene desaturase (en- coded by crtQ (zds)). The pdsⴚ and zdsⴚ strains synthe- size phytoene and ?-carotene, respectively, both of which absorb to shorter wavelength than ␤-carotene. Compared with wild type, the mutant A1 ⴚ(FeS) ⴚ A1(FeS)ⴚ difference spectra, measured in cells and pho- tosystem I complexes, retain the electrochromic band shift centered at 450 nm but show a complete loss of the electrochromic band shifts centered at 500–510 nm. Thus, the latter clearly arise from ␤-carotene. In the wild type, the electrochromic band shift of the slow phase (centered at 500 nm) is shifted by 6 nm to the blue compared with the fast phase (centered at 506 nm). Thus, the carotenoid pigments acting as electrochromic markers during the fast and slow phases of A1 ⴚ oxida- tion are different, indicating the involvement of both the PsaA- and the PsaB-side phylloquinones in photo- system I electron transport.