Pathways of proton release in the bacteriorhodopsin photocycle.
The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.
Zimányi
L
L
Department of Physiology and Biophysics, University of California, Irvine 92717.
Váró
G
G
Chang
M
M
Ni
B
B
Needleman
R
R
Lanyi
J K
JK
D001436:Bacteriorhodopsins / Q000378:metabolism* / Q000528:radiation effects*; D001692:Biological Transport; D019617:Halobacterium salinarum / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016623:Ion Pumps; D007700:Kinetics; D008956:Models, Chemical; D010775:Photic Stimulation; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects; D013053:Spectrophotometry
D016623:Ion Pumps; D011522:Protons; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins