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 O2. A tetranuclear manganese cluster associated with integral membrane subunits of PSII catalyzes water oxidation. The 33-kDa water-soluble PSII subunit, or manganese-stabilizing protein (MSP), stabilizes the O2-evolving manganese cluster and accelerates O2 evolution. Spinach PSII can be depleted of native MSP under conditions which retain a functional manganese cluster. Reconstition 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 that [V235A]-MSP was destabilized by low temperature to a greater extent than the wild-type protein. However, the effect of temperature on the secondary structure of both the mutant and wild-type proteins was small compared to the temperature-independent destabilization of secondary structure induced by the mutation. These results demonstrate that the V235A mutation introduces an activation energy barrier for assembly of MSP into PSII, and it is suggested that the mutation acts by inhibiting isomerization of one or more prolyl peptide bonds required for assembly.
Betts
S D
SD
Department of Biology, University of Michigan, Ann Arbor 48109-1048.
Ross
J R
JR
Pichersky
E
E
Yocum
C F
CF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003080:Cold Temperature; D046911:Macromolecular Substances; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D045332:Photosystem II Protein Complex; D017354:Point Mutation; D017510:Protein Folding; D011506:Proteins; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D008667:Metalloproteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011506:Proteins; C064388:oxygen-evolving complex, 33 kDa protein; C054266:photosystem II manganese-stabilizing protein; D008345:Manganese