An investigation of the PsbS protein isolated from spinach chloroplast membranes.

Dissipation of excess light energy in plant photosynthetic membranes plays an
important role in the response of plants to the environment, providing short-term
balancing between the intensity of sunlight and photosynthetic capacity. The carotenoid
zeaxanthin and the photosystem Il subunit PsbS play vital roles in this process, but the
mechanism of their action is largely unexplained. This thesis reports a novel procedure
for the extraction of the PsbS protein from spinach thylakoids, including a detailed
account of the developmental process and characterisation of the isolated protein. The
ability of the PsbS protein to bind xanthophyll cycle carotenoids in vitro was assessed,
leading to the observation that the isolated protein was able to bind exogenous
zeaxanthin, the binding resulting in a strong red shift in the absorption spectrum, and
the appearance of characteristic features in the resonance Raman spectrum and a distinct
circular dichroism spectrum, indicating pigment-protein, as well as specific pigmentpigment,
interaction. A strong shift in the absorption spectrum of PsbS phenylalanine
residues after zeaxanthin binding was observed. It is concluded that zeaxanthin binding
to PsbS is the origin of the well known energy dissipation-related 535-nm absorption
change. The ability of this PsbS-zeaxanthin complex to affect the rate of chlorophyll
fluorescence quenching of the major LHcn antenna protein is detailed, revealing an
increase in the rate of quenching, whilst the magnitude of quenching remained constant.
The altered properties of zeaxanthin and PsbS after in vitro reconstitution and their
subsequent effect on LHCnb provide the first direct indication about how they regulate
energy dissipation.