Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties.
The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.
Wyman
Aaron J
AJ
Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
Popelkova
Hana
H
Yocum
Charles F
CF
D002942:Circular Dichroism; D017124:Conserved Sequence / Q000235:genetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D045332:Photosystem II Protein Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D018724:Spinacia oleracea; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D010446:Peptide Fragments; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; C052389:oxygen-evolving enhancer protein 1, plant; C054266:photosystem II manganese-stabilizing protein; D014443:Tyrosine; D008345:Manganese; D010649:Phenylalanine; D014364:Tryptophan