Photoaccumulation of two ascorbyl free radicals per photosystem I at 200 K

Illumination of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 at 200 K in the presence of ascorbate leads to the formation of two ascorbyl radicals per PSI, which are formed by P700(+) reduction by ascorbate. During photoaccumulation, one half of the ascorbyl radicals is formed with a halftime of 1 min and the other half with a halftime of 7 min. Pulsed electron paramagnetic resonance (EPR) experiments with protonated/deuterated PSI show that a PSI proton/deuteron is strongly coupled to the ascorbyl radical. Our data indicate that reactive ascorbate molecules bind to PSI at two specific locations, which might be symmetrically located with respect to the pseudo-C(2) axis of symmetry of the heterodimeric core of PSI. Reduction of P700(+) by ascorbate leads to multiple turnover of PSI photochemistry, resulting in partial photoaccumulation of the doubly reduced species (F(A)(-), F(B)(-)). A modified form of F(B)(-)-in accordance with Chamorovsky and Cammack [Biochim. Biophys. Acta 679 (1982) 146-155], but not of F(A)(-), is observed by EPR after illumination at 200 K, which indicates that reduction of F(B) at 200 K is followed by some relaxation process, in line with this cluster being the most exposed to the solvent.