The role of the retinylidene Schiff base counterion in rhodopsin in determining wavelength absorbance and Schiff base pKa.
Glu-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Purified mutant rhodopsin pigments were prepared in which Glu-113 was replaced individually by Gln (E113Q), Asp (E113D), Asn (E113N), or Ala (E113A). E113Q, E113N, and E113A existed as pH-dependent equilibrium mixtures of unprotonated and protonated Schiff base (PSB) forms. The Schiff base pKa values determined by spectrophotometric titration were 6.00 (E113Q), 6.71 (E113N), and 5.70 (E113A). Thus, mutation of Glu-113 markedly reduced the Schiff base pKa. The addition of NaCl promoted the formation of a PSB in E113Q and E113A. An exogenously supplied solute anion replaced Glu-113 to compensate for the positive charge of the PSB in these mutants. The lambda max values of the PSB forms of the mutants in NaCl were 496 nm (E113Q), 506 nm (E113A), 510 nm (E113D), and 520 nm (E113N). To evaluate the effect of different types of solute anions on lambda max values, mutants were prepared in sodium salts of halides, perchlorate, and a series of carboxylic acids of various sizes and acidity. The lambda max values of E113Q and E113A depended on the solute anion present and ranged from 488 nm to 522 nm for E113Q and from 486 nm to 528 nm for E113A. The solute anion affected the lambda max values of E113N and E113D to lesser degrees. The reactivities of the mutants to hydroxylamine were also studied. Whereas rhodopsin was stable to hydroxylamine in the dark, E113N reacted slowly and E113Q reacted rapidly under these conditions, indicating structural differences in the Schiff base environments. The lambda max values and solute anion dependencies of the Glu-113 mutants indicate that interactions between Schiff base and its counterion play a significant role in determining the lambda max of rhodopsin.
Sakmar
T P
TP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Franke
R R
RR
Khorana
H G
HG
D000818:Animals; D000838:Anions; D001965:Bromides / Q000737:chemistry; D002417:Cattle; D002712:Chlorides; D004252:DNA Mutational Analysis; D005459:Fluorides / Q000737:chemistry; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D066298:In Vitro Techniques; D007454:Iodides / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry*; D012545:Schiff Bases / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D014162:Transfection
D000838:Anions; D001965:Bromides; D002712:Chlorides; D005971:Glutamates; D006898:Hydroxylamines; D007454:Iodides; D012545:Schiff Bases; D019811:Hydroxylamine; D012243:Rhodopsin; D005459:Fluorides