The Effect of Different Metal Cation Binding on the Proton Pumping in Bacteriorhodopsin

Abstract

The first section of this paper is a detailed summary of studies made by us and others on metal cations binding to deionized bacteriorhodopsin (dIbR) and its variants. Our studies include the luminescence experiments of Eu 3 + binding to dIbR and potentiometric studies of Ca" binding to dIbR, to deionized bR mutants, to bacterioopsin, and to dIbR with its C-terminus removed. The results suggest the presence of two classes of binding sites, one class has two high-affinity constants, and one has one low-affinity constant. For Ca" binding, there is one metal cation in each of the two high-affinity sites which are coupled to the charged aspartates 85 and 212 (known to be in the retinal cavity) but not coupled to each other. The lowaffinity class can accommodate 0-6 Ca" ions and most of them are bound to the surface. Mg" has a slightly smaller value for its binding constant to the highestaffinity site. Thus, one expects more Ca" than Mg" bound to the two high-affinity sites. In the second section, we summarize our recent study on the effect of metal cation charge density (Ca'", Mg", Eu 3 +, Tb 3 +, H03+, Dy3+) on the kinetics of both Schiff base deprotonation and proton transport to the extracellular surface. For all metal cations, the apparent rate constant of the slow components of the deprotonation process is the same as that for the transport process at 22°C. The temperature studies, however, show this apparent equality to be fortuitous and to result from cancellation of the contribution of the energy and entropy of activation. Thus, while the entropy of activation is positive for the deprotonation process, it is negative for the proton transport process. These kinetic parameters depend weakly on the charge density, but in an opposite sense for the two processes. These results suggest that the deprotonation is not the rate-limiting step for the proton transport process. A possible mechanism is proposed in which a hydrated metal cation is used to induce the deprotonation of the protonated Schiff base and to dissociate one of its HzO molecules to donate the proton in the L~M process.b~-:>(bR)*~I460~J625~~IO~L550~M4l2~N~b- H+ +H+In the L sso~M412 step, a proton is transferred from

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

“…There are two proposals regarding binding of cations to bacteriorhodopsin affecting the protonation state of Asp-85. The first proposal regards the cations binding to the hydrophilic side groups within the protein (acids or alcohols) and perhaps trapped water, and there is an equilibrium between protonated and cation-bound states dependent on the pK a of the group(s) (18,38,39). Perhaps it is binding to one or more of the groups by the cation that controls the Asp-85 protonation state.…”

The titrations of Asp‐85 and of the cation binding residues in bacteriorhodopsin are not coupled

“…The purpleIblue transition is closely related to the binding of 8^10 metal cations (Ca P and Mg P ) to bR (see [12] for a recent review of metal cation binding to bacteriorhodopsin).…”

Eu3+ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

“…It has been postulated that the role of cations in the native bR is only to regulate the surface pH by shielding the negative lipidic charges, thereby indirectly stabilizing the deprotonated Asp85 [27,28]. Since 75% delipidation and monomerization still retains the ability of three Eu 3+ cations to remain bound to the protein, this suggests binding directly to the protein side groups, as previously discussed [13,15,16]. While these experiments cannot determine exactly where in the protein the cations are bound (or even if Eu 3+ occupies the same sites as the native Ca 2+ and Mg 2+ ), these results lend support to a direct protein binding model in which their binding must be controlled by the p K a of the side group binding sites.…”

“…All spectroscopic and biochemical tests suggest that the blue membrane formed by each method is the same species [14,15]. Furthermore, the proton pumping function of bR is halted upon deionization but can be recovered by addition of a number of different cations to the blue bR [13–16]. This illustrates the importance of cations in the function.…”

“…This blue bR can be formed by passing through a deionizing column [12], removal of cations by EDTA chelation [13] or by acidifying the solution [11]. All spectroscopic and biochemical tests suggest that the blue membrane formed by each method is the same species [14,15]. Furthermore, the proton pumping function of bR is halted upon deionization but can be recovered by addition of a number of different cations to the blue bR [13–16].…”