Replacement of aspartic residues 85, 96, 115, or 212 affects the quantum yield and kinetics of proton release and uptake by bacteriorhodopsin (site-specific mutagenesis/proton pump/differential conductivity/membrane protein/interfacial charge transfer)

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 bacterio- rhodopsin. For the Asp-96 -. Asn mutant, proton uptake was slowed by >1 order of magnitude with no observable effect on the release step. For Asp-85 -. Asn, HI 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 substi- tutions 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 substitu- tions affect the kinetics of either HI release or HI uptake, but not both. This implies that the two steps occur independently of each other after initiation of the photocycle.