MULTIFREQUENCY CROSS-CORRELATION PHASE FLUOROMETRY OF CHLOROPHYLL-A FLUORESCENCE IN THYLAKOID AND PSII-ENRICHED MEMBRANES

We present here a comparative study on the decay of chlorophyll (Chl) a fluorescence yield in thylakoid membranes and photosystem II (PSII)- enriched samples, measured with multifrequency cross-correlation phase fluorometry. These measurements confirm the general conclusions of Va n Mieghem et al. (Biochim. Biophys. Acta 1100, 198-206, 1992), obtaine d with a flash method, on the effects of reduction of the primary quin one acceptor (Q(A)) on Chl a fluorescence yield of PSII. Different sta tes of the reaction centers of PSII were produced by: (1) pretreatment with sodium dithionite and methyl viologen followed by laser illumina tion: the doubly reduced Q(A) (Q(A)H-2) centers; (2) with laser illumi nation or pretreatment with diuron: Q(A)- centers; and (3) the additio n of micromolar concentration of dichlorobenzoquinone (DCBQ): oxidized Q(A) centers. The data were analyzed with Lorentzian distribution as well as with multiexponential fluorescence decay functions. The analys is with Lorentzian distribution function showed that upon formation Of Q(A)-, the major lifetime distribution peak shifted to longer lifetim es: from 0.25 ns to 1.66 ns (pea thylakoid membranes) and from 0.24 ns to 1.31 ns (core PSII). However, when Q(A)H-2 was formed, the lifetim e distribution peaks shifted back to shorter lifetimes (0.57-0.77 ns) both in thylakoids and PSII membranes. Multiexponential analysis showe d three lifetime components: fast (40-400 ps), middle (300-1500 ps) an d slow (5-25 ns). When Q(A)- was formed in PSII centers, the amplitude of the fast component decreased, but both the amplitude and the lifet ime of the middle component increased severalfold. However, when Q(A) was doubly reduced, the amplitude of the fast component increased and the amplitude of the middle component decreased; in addition, the life time of the slow component increased. All of the above results are con sistent with the conclusions that PSII charge separation is decreased when Q(A)- is formed and increased when doubly reduced Q(A) is formed.