Millisecond time-resolved EPR of the spin-polarised triplet in the isolated Photosystem II reaction centre

Abstract

The D 1/D 2-cytochrome b-559 PS II reaction centre was isolated using Triton X-100, and this detergent was subsequently exchanged for dodecyl β-maltoside. This preparation is stable in the dark, and at cryogenic temperatures in the light. The isolated PS II reaction centre showed a steady-stage Δm = 1 EPR spectrum of a chlorophyll triplet state, at temperatures under 20 K. Its polarisation pattern was AEEAAE, characteristic of the molecular triplet state of the primary donor ( 3P680) populated exclusively in the T 0 level via the 3RP. Increasing the temperature within the range 5–20 K caused a decrease in EPR signal intensity (the z peak being initially more sensitive than the x or y peaks), but the polarisation pattern remained unchanged. The millisecond time-resolved response of the intensity of the x, y and z peaks to a light excitation step pulse was measured, and the light-on and -off responses could all be described by a single exponential at low (≤ 30 μW) microwave power and low (≤ 100 mW) light intensity. The values of the decay rate constants, k x , k y and k z , extrapolated to zero microwave power, were compared with values reported for PS II preparations of larger antenna size. The value for k x , 605 s −1, was distinctly shorter than those previously reported, and tended towards that reported for biligated monomeric Chl a in methyltetrahydrofuran. k x was found to be extremely sensitive to microwave power. The value for k z was 185 s −1, and that for k y 965 s −1. A second exponential component was seen in the kinetics of the y and z peaks at high microwave power due to a non-negligible microwave-induced population of the T ±1 levels. The decay rate constants were all independent of the wavelength (458 or 514 nm) and intensity (50–300 mW) of the excitation light. They remained unchanged when the steady-state EPR signal intensity was reduced by 50% by increasing the temperature. This was taken to indicate that the effect of temperature was on the populating process. A theoretical model has been developed which can explain most of the effects of microwave power on the EPR kinetics.