The present study for the first time describes a close relationship between a change in the states of Cyt b559, a damage to Mn complex and a rapid reduction of tyrosine D (Y(D)) as a function of temperature in spinach thylakoid membranes. Measurements of the EPR signal of dark stable tyrosine D in heat-treated thylakoid membranes showed a gradual decay of the oxidized state of tyrosine D with the progression of temperature. Simultaneously, it leads to the conversion of high-potential Cytochrome b559 into its low-potential form. We have speculated a possible involvement of Cytochrome b559 in the primary reduction events of tyrosine D in dark at high temperature. However, rapid reduction of tyrosine D may also be due to the disassembly of the Mn clock, which causes exposure of Y(D) to the lumen and thereby its reduction by some unknown factor. These conclusions are supported by the measurements of Mn(2+) release and thermoluminescence curves of various charge pairs in heat-treated thylakoid membranes. The results reveal an important aspect on the role of Cyt b559 in PS II during temperature stress.