Hanley, Jonathan Antony; (1993) Characterisation of the photosystem-I reaction centre complex. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis reports studies of the structure and function of higher plant photosystem-I electron transfer components. The function of stromal side polypeptides has also been investigated with particular reference to the transfer of electrons through the bound iron-sulphur centres (Fe-SA/B) to NADP+. The principle techniques used have been low temperature electron paramagnetic resonance spectroscopy (EPR), room temperature kinetic spectroscopy and column chromatography. First reported is the isolation of a photosystem-I core complex devoid of its terminal bound iron-sulphur centres and other peripheral polypeptides. The core complex was prepared by incubation of either Triton or Digitonin photosystem-I in 6.8M urea. EPR analysis reveals it to contain P700, and A0 and A1 room temperature P700+ re-reduction kinetics indicate the presence of Fe-Sx. The respective role of PSI-C, the Fe-SA/B holoprotein, PSI-D and PSI- E was investigated by carrying out a series of reconstitution experiments utilising the PS-I core complex and stromal polypeptides isolated from photosystem-I by n-butanol extraction. Room temperature kinetic experiments demonstrated that reconstitution of the core complex with the PSI-C holoprotein resulted in the recovery of 20 ms kinetics observed in control PS-I. EPR analysis showed that PSI-D, and possibly PSI-E, was also required in order stabilise the rebound PSI-C holoprotein. Measurement of the rate of NADP+ photoreduction, by monitoring change in OD 340 nm following 30 second periods of illumination with saturating white light, demonstrated that redox centres Fe-SA/B were essential for forward electron transfer to NADP+ at room temperature. The reconstitution experiments also showed that components PSI-D and PSI-E were essential for interaction with the soluble intermediates ferredoxin and ferredoxin NADP oxidoreductase. Illumination of photosystem-I at 4°C in the presence of sodium dithionite has been shown to result in the double reduction of the quinone species A1 thereby rendering it EPR silent. This thesis reports that gradual quinone double reduction also results in a concomitant disappearance of the photo-accumulated EPR signal previously attributed to A1-. The assignment of the g = 2.0048 and 0.95 mT linewidth EPR signal, photo-accumulated by 205K illumination, to the reduction of the quinone component was thus confirmed. Photo-accumulation of A1- and A0- by illumination of photosystem-I at 230K in the presence of sodium dithionite at pH 10.0 resulted in the rise of approximately 4 spins per P700+ reaction centre. This forms the first direct evidence that the photosystem-I reaction centre complex may exhibit the same C2 symmetry observed in other types of photosynthetic reaction centre complex.

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