Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.