New insights on Chl _D1 function in Photosystem II from site-directed mutants of D1/T179 in Thermosynechococcus elongatus

The monomeric chlorophyll, Chl _D1 , which is located between the P _D1 P _D2 chlorophyll pair and the pheophytin, Pheo _D1 is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg ²⁺ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn ₄ CaO ₅ cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *Chl _D1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of P _D1 P _D2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the Chl _D1 ⁺ Pheo _D1 ⁻ radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of ¹ O ₂ generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to Chl _D1 upon Q _A ⁻ formation, indicates that in the T179H-PSII and in the WT*3-PSII, the Chl _D1 itself is the chlorophyll that is first damaged by ¹ O ₂ , whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, Chl _D1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of Chl _D1 very likely contribute to the well-known electrochromic shifts observed at ~430 nm during the S-state cycle.