Biochemical and Spectroscopic Properties of Heat-Treated Manganese Stabilizing Protein

Three water soluble proteins with molecular masses of 17, 23 and 33 kDa, located on the lumenal side of photosystem 2 (PSII), are responsible for the stability and the optimum function of the inorganic cofactors of the oxygen evolving complex (OEC) (1). More specifically, 17 and 23 kDa proteins are responsible for retention of Ca2+ and Cl− whereas a 33 kDa protein, called Manganese Stabilizing Protein (MSP), is responsible for the integrity and optimum function of the manganese cluster of OEC (1,2). Protein digestion and cross linking studies have identified the E loops of the intrinsic, chlorophyll-a binding proteins CP47 and CP43 as binding sites of MSP (3–5). Cyt b559 and a small intrinsic protein (<10 kDa) have also been proposed to provide ligands for the protein (5,6). Biochemical studies have shown that there are two copies of MSP per PSII reaction center (7,8). Protein digestion and site-directed mutagenesis have identified the N- terminus and the C- terminus of MSP as responsible for binding to CP47 and for maintaining structural features in solution necessary for functional binding, respectively (9, Betts et al, unpublished results). Here we report the biochemical and spectroscopic properties of native and recombinant MSP incubated at either room temperature or at 90°C. We show that heated MSP retains a remarkable ability to rebind to PSII and reactivate O2 evolution after long incubation at high temperatures. The secondary and tertiary structures of both species were examined by far-UV circular dichroism (CD), near-UV CD and size-exclusion chromatography. Cooling of heat-treated MSP results in refolding to a nearly native conformation. Structural and functional thermostability, along with other properties of MSP, suggest that it belongs to a family of “natively unfolded” proteins that participate in protein-protein interactions (10).