Correlation between flash-induced oxygen evolution and fluorescence yield kinetics in the 0 to 16 μs range in Chlorella pyrenoidosa during incubation with hydroxylamine

Abstract

Following flash excitation, oxygen pulses and fluorescence kinetics in the time range 0–16 μs were studied in the alga Chlorella pyrenoidosa during incubation with various concentrations of hydroxylamine. The obtained results could be explained considering four effects of hydroxylamine. 1. 1. Hydroxylamine removes (reduces) oxidizing equivalents, generated in the water-splitting system by flash excitation. This process does not markedly affect the fluorescence yield kinetics between 0 and 16 μs following the ignition of a flash and reaches a constant rate within a few minutes, but possibly within a few seconds, after addition of hydroxylamine. In a sequence of flashes separated by dark time t d, the steady-state oxygen yield in the flashes is exp(− kt d), the yield at t d = 0 being taken equal to 1, where k = (0.1 + β[NH 2OH])s −1, with [NH 2OH] in mM and β = 0.6 mM −1, provided [NH 2OH] ⩾ 0.5 mM. 2. 2. An inhibition between Z, the physiological donor and the oxidized reaction center pigment P + occurs, proceeding as exp(− k i t i ) where t i is the incubation time with hydroxylamine and k i = ( α[NH 2OH]) min −1, with [NH 2OH] in mM and α = 0.14 mM −1. This process not only inhibits oxygen evolution capability, but also decreases the amplitude of the fluorescence yield difference ΔФ = Ф(16 μ s) − Ф(2 μ s) induced by a flash in the steady state. In a fraction of the reaction centers this inhibition occurs “immediately” after the addition of hydroxylamine. These observations, combined with the conclusion of Cheniae and Martin (1971, Plant Physiol. 47, 568–575) that the inhibition of the Hill reaction is related to the extraction of bound maganese, indicate that the reaction between Z and P + requires bound manganese. 3. 3. In the inhibited centers a second donor for P +, D, connected to an entry site for the artificial electron donor hydroxylamine becomes apparent. 4. 4. A flash-induced oxygen uptake signal was observed in the presence of hydroxylamine, which was shown to be caused by a system II reaction. The effects under (1) and (4) were reversed in the dark if hydroxylamine was removed by washing. The effects under (2) and (3) were reversed during illumination of a washed sample.