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• pH Dependence of Light-Driven Proton Pumping by an Archaerho...

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• Excitation Energy-Transfer and the Relative Orientation of R...

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Abstract

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 KCl (compared to ∼2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pK_a of E204 decreases to 8.1±0.1 (from ∼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 ∼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 KCl), 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 (∼30%) undergoes a conversion into a shorter wavelength species, P480, with pK_a ≈ 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.