Binding of a single divalent cation directly correlates with the blue-to-purple transition in bacteriorhodopsin.

Abstract

We have characterized a unique divalent cation binding site on bacteriorhodopsin which controls the blue-to-purple transition in the purple membrane of Halobacterium halobium. To identify this site we first showed the correlation between the binding of one Ca2+ per bacteriorhodopsin and the amount of blue membrane converted to purple membrane. When the free Ca2+ was reduced below 1 pM, and the pH was set below 5.0 with 0.5 mM citrate, only binding to this high-affinity site was observed, and we could separate its effect from the effect of other divalent cations binding to the membrane under other conditions. Second, the titration ofpurple membrane showed that protons are taken up in two distinct steps, about 13 with a pK. of 4-5 and an additional 2 protons with a pK. of 2.75, in 5 mM MgSO4. The latter is identical to the pK. for the purple-to-blue transition in 5 mM MgSO4. Taken together, these observations strongly suggest a direct role for cations in the regulation of the bacteriorhodopsin color under normal conditions. We have also found that the intrinsic pK. for the purple-to-blue transition is about 2.05, suggesting this is the pK. of the group or groups that, when protonated, lead to the blue membrane. Previously published data can now be interpreted to suggest that the cation regulates an active site near the retinal chromophore. A binding site for the divalent cation that includes Asp-212 and interactions with the protonated Schiff base, Asp-85, Tyr-57, Tyr-185, and Arg-82 is proposed.

PURPLE MEMBRANE: SURFACE CHARGE DENSITY and THE MULTIPLE EFFECT OF pH and CATIONS

“…They suggested that this value was too high (especially at pH 1) and was probably due to complications from protein conformational changes. Jonas and Ebrey (1990) have repeated Kimura et al's experiment, mostly at higher salt concentrations, and their results suggest a higher intrinsic pK, (ca 2) and a much lower surface charge density, from less than 1, to 3 charges per 1140 A' . A constant surface charge density could not be obtained from this data at the different ionic strengths.…”

“…Those experimental results which are probably specific for the N-terminal, extracellular side of the membrane are mostly between 1 and 4 negative charges per bR. At low pH, the acid-induced purple-to-blue transition as a function of the ionic strength suggests surface charge densities ranging from less than 1, to 3 negative charges per bR at pH 2 (Jonas and Ebrey, 1990). At pH 7.5, Ehrenberg et al (1989) found 2 negative charges per bR.…”

“…Asp-85 has been suggested to act as a counterion to the protonated Schiff base Otto et al, 1990;Subramaniam et al, 1990), and its protonation probably leads to the formation of the blue membrane (see below). Jonas and Ebrey (1990) have proposed that the removal of a divalent cation from the bR active site Figure 1. The structure of the purple membrane.…”

“…In these models the cations influence the local pH at a protonateable, color regulating group, such as the Schiff base counterion. Cation effects on the local pH are no doubt present and important, in addition to the possibility of a specific cation helping to regulate the ionization state of the counterion (Jonas and Ebrey, 1990). An alternative explanation for the influence of local pH has been that it causes a conformational change in the protein that indirectly regulates the color of the membrane, possibly by moving the counterion farther away from the Schiff base (e.g.…”

The binding site of the strongly bound Eu3+ in Eu3+‐regenerated bacteriorhodopsin

“…More recent work by Ebrey etal. [24] on the correlation of color change and the number of Ca ~+ ions bound and the H + ions produced, strongly suggested the importance of one calcium ion in bR. From the recently proposed model for the structure of bR [25], Ebrey suggested that tt~is Ca a+ ion might be located within the retinal pocket [24].…”