COLD-SENSITIVE ASSEMBLY OF A MUTANT MANGANESE-STABILIZING PROTEIN CAUSED BY A VAL TO ALA REPLACEMENT

Photosystem II (PSII) is a multisubunit transmembrane protein complex that oxidizes water and evolves O-2. A tetranuclear manganese cluster associated with integral membrane subunits of PSII catalyzes water oxi dation. The 33-kDa water-soluble PSII subunit, or manganese-stabilizin g protein (MSP), stabilizes the O-2-evolving manganese cluster and acc elerates O-2 evolution. Spinach PSII can be depleted of native MSP und er conditions which retain a functional manganese cluster. Reconstitio n of MSP-depleted PSII with recombinant MSP was equally efficient at 4 and 22 degrees C. Replacement of Val235 (a conserved residue near the C-terminus of MSP) with Ala inhibited assembly of MSP at 4 degrees C, but not at 22 degrees C. Once assembled, [V235A]MSP remained bound to PSII even at 4 degrees C and in the presence of low concentrations of urea. Results from far-UV circular dichroism spectrometry indicated t hat [V235A]-MSP was destabilized by low temperature to a greater exten t than the wild-type protein. However, the effect of temperature on th e secondary structure of both the mutant and wild-type proteins was sm all compared to the temperature-independent destabilization of seconda ry structure induced by the mutation. These results demonstrate that t he V235A mutation introduces an activation energy barrier for assembly of MSP into PSII, and it is suggested that the mutation acts by inhib iting isomerization of one or more prolyl peptide bonds required for a ssembly.