Leucine 245 is a critical residue for folding and function of the manganese stabilizing protein of photosystem II

Ln solution, Manganese Stabilizing Protein, the polypeptide which is respon sible for the structural and functional integrity of the manganese cluster in photosystem II, is a natively unfolded protein with a prolate ellipsoid shape [Lydakis-Simantiris et al. (1999) Biochemistry 38, 404-414; Zubrzycki et al. (1998) Biochemistry 37, 13553-13558]. The C-terminal tripeptide of Manganese Stabilizing Protein was shown to be critical for binding to photo system II and restoration of O-2 evolution activity [Betts et al. (1998) Bi ochemistry 37, 14230-14236]. Here, we report new biochemical, hydrodynamic, and spectroscopic data on mutants E246K, E246STOP, L245E, L245STOP, and Q2 44STOP. Truncation of the final dipeptide (E246STOP) or substitution of Glu 246 with Lys resulted in no significant changes in secondary and tertiary s tructures of Manganese Stabilizing Protein as monitored by CD spectroscopy. The apparent molecular mass of the protein remained unchanged, both mutant s were able to rebind to photosystem II, and both proteins reactivate Oz ev olution. Manganese Stabilizing Protein lacking the final tripeptide (L245ST OP), or substitution of Glu for Leu245 dramatically modified the protein's solution structure. The apparent molecular masses of these mutants increase d significantly, which might indicate unfolding of the protein in solution. This was verified by CD spectroscopy. Both mutant proteins rebound to phot osystem II with lower affinities, and activation of Oz evolution was decrea sed dramatically. Enhancement of these defects was observed upon removal of the final tetrapeptide (Q244STOP). These results indicate that Leu245 is e ssential to maintaining Manganese Stabilizing Protein's solution structure in a conformation that promotes efficient binding to photosystem II and/or for the subsequent steps that lead to enzyme activation. Based on an analys is of the properties of C-terminal mutations, a hypothesis for structural r equirements for functional binding of Manganese Stabilizing Protein to phot osystem II is presented. Effects of C-terminal mutations on the UV spectrum of Manganese Stabilizing Protein were also examined. Mutations that alter solution structure also affect a 293 nm absorption shoulder which is assign ed to the only tryptophan residue, Trp241, in the protein, and this absorba nce feature is shown to be a useful indicator of alterations to the Trp241 environment.