Replacement of aspartic residues 85, 96, 115, or 212 affects the quantum yield and kinetics of proton release and uptake by bacteriorhodopsin.
Recently, a number of aspartic acid mutants of bacteriorhodopsin have been shown to be defective in steady-state proton transport. Here we report time-resolved measurements of light-induced proton release and uptake for these mutants. Proton transfers between the protein and the aqueous phase were directly monitored by measuring changes in the bulk conductivity of a micellar solution of bacteriorhodopsin. For the Asp-96----Asn mutant, proton uptake was slowed by greater than 1 order of magnitude with no observable effect on the release step. For Asp-85----Asn, H+ uptake occurred with normal kinetics, but the yield was significantly lower compared with either the Asp-96----Asn mutant or wild type, especially at pH 6. Substitution of glutamate for Asp-85 or Asp-96 had smaller but detectable effects on the kinetics and quantum yield of proton movements. Both asparagine and glutamate substitutions of aspartates at positions 115 and 212 lowered the proton quantum yields. Of these, only the Asp-115----Asn mutant showed an effect on the proton release step, and only the Asp-212----Glu mutation decreased the proton uptake rate. These experiments imply an obligatory role for Asp-96 in H+ uptake in the normal operation of the bacteriorhodopsin proton pump. The results also indicate that the amino acid substitutions affect the kinetics of either H+ release or H+ uptake, but not both. This implies that the two steps occur independently of each other after initiation of the photocycle.
Marinetti
T
T
Biophysics Laboratory, Rockefeller University, New York, NY 10021.
Subramaniam
S
S
Mogi
T
T
Marti
T
T
Khorana
H G
HG
D001216:Asparagine; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000235:genetics* / Q000378:metabolism; D001693:Biological Transport, Active; D005971:Glutamates; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D009154:Mutation; D011522:Protons
D005971:Glutamates; D011522:Protons; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine