Mutation of a surface residue, lysine-129, reverses the order of proton release and uptake in bacteriorhodopsin; Guanidine hydrochloride restores it

K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of bacteriorhodopsin. Replacement of K129 with a histidine alters the pK(a)'s of two key residues in the proton transport pathway, D85, and the proton release group (probably E204); the resulting pigment has properties that differ markedly from the wild type. 1) In the unphotolyzed state of the K129H mutant, the pK(a) of D85 is 5.1 +/- 0.1 in 150 mM KCI (compared to similar to 2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pK(a) of E204 decreases to 8.1 +/- 0.1 (from similar to 9.5 in the wild-type pigment). 2) The pK(a) of E204 in the M state is 7.0 +/- 0.1 in K129H, compared to similar to 5.8 in the wild-type pigment. 3) As a result of the change in the pK(a) of E204 in M, the order of light-induced proton release and uptake exhibits a dependence on pH in K129H differing from that of the wild type: at neutral pH and moderate salt concentrations (150 mM KCI), light-induced proton uptake precedes proton release, whereas it follows proton release at higher pH. This pumping behavior is similar to that seen in a related bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129. 4) At alkaline pH, a substantial fraction of all-trans K129H pigment (similar to 30%) undergoes a conversion into a shorter wavelength species, P480, with pK(a) approximate to 8.1, close to the pK(a) of E204. 5) Guanidine hydrochloride lowers the pK(a)'s of D85 and E204 in the ground state and the pK(a) of E204 in the M intermediate, and restores the normal order of proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and E204 is weaker than in wild-type bacteriorhodopsin. In the unphotolyzed pigment, the change in the pK(a)'s of either residue when the other changes its protonation state is only 1.5 units compared to 4.9 units in wild-type bacteriorhodopsin. In the M state of photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild-type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network that couples E204 and D85. Substitution of K129 with a histidine residue causes structural changes that alter this coupling and affect the pK(a)'s of E204 and D85.