Effects of tryptophan mutation on the deprotonation and reprotonation kinetics of the Schiff base during the photocycle of bacteriorhodopsin.
The rates of deprotonation and reprotonation of the protonated Schiff base (PSB) are determined during the photocycle of nine bacteriorhodopsin mutants in which Trp-10, 12, 80, 86, 137, 138, 182 and 189 are individually substituted by either phenylalanine or cysteine. Of all the mutants, the replacement of Trp-86, Trp-182, and Trp-189 by phenylalanine and Trp-137 by cysteine is found to significantly alter the rate of the deprotonation, but not that of the reprotonation process. As compared with ebR, the Trp-86 mutation dramatically increases the rate of deprotonation of the PSB while the Trp-182 mutation greatly decreases this rate. Temperature dependence studies on the rate constants of the deprotonation demonstrate that the different energetic and entropic effects of the mutation are responsible for the observed different kinetic behavior of the Trp-86 and Trp-182 mutants as compared with that of ebR. In the case of Trp-86 mutant, a large decrease in both energy and entropy of activation suggests that the mutation of this tryptophan residue opens up the protein structure as a result of eliminating the hydrogen-bonding group on its side chain by a phenylalanine substitution. A correlation is observed between the proton pumping yield and the relative amplitudes of the slow deprotonation component but not with rate constants of the rise or decay process at constant pH. These results are best discussed in terms of the heterogeneity model (with parallel cycle) rather than back reaction model.
Wu
S
S
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Chang
Y
Y
el-Sayed
M A
MA
Marti
T
T
Mogi
T
T
Khorana
H G
HG
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D011522:Protons; D012545:Schiff Bases; D001436:Bacteriorhodopsins; D014364:Tryptophan