DETERMINATION OF THE TRANSIENTLY LOWERED PK(A) OF THE RETINAL SCHIFF-BASE DURING THE PHOTOCYCLE OF BACTERIORHODOPSIN

Reprotonation of the transiently deprotonated retinal Schiff base in t he bacteriorhodopsin photocycle is greatly slowed when the proton dono r Asp-96 is removed with site-specific mutagenesis, but its rate is re stored upon adding azide or other weak acids such as formate and cyana te. As expected, between pH 3 and 7 the rate of Schiff base protonatio n in the photocycle of the D96N mutant correlates with the concentrati ons of the acid forms of these agents. Dissection of the rates in the biexponential reprotonation kinetics of the Schiff base between pH 7 a nd 9 yielded calculated rate constants for the protonation equilibrium . Their dependencies on pH and azide or cyanate concentrations are con sistent with both earlier suggested mechanisms: (i) azide and other we ak acids may function as proton carriers in the protonation equilibriu m of the Schiff base, or (ii) the binding of their anionic forms may c atalyze proton conduction to and from the Schiff base. The measured ra te constants allow the calculation of the pK(a) of the Schiff base dur ing its reprotonation in the photocycle of D96N. It is 8.2-8.3, a valu e much below the pK(a) determined earlier in unphotolyzed bacteriorhod opsin.