Influence of the charge at D85 on the initial steps in the photocycle of bacteriorhodopsin

Abstract

Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.

Figure 1

Detailed view of the environment around the protonated Schiff bases in BR (red) and HR (green). The two structures were overlaid to minimize the root mean-square of the Ca atoms of the total proteins. For clarity, only part of helices C and G are shown, as well as the side chains of D85 and R82 (nomenclature of BR) and T111 (HR), and D212 and K216 of BR, with their corresponding residues in HR. The chloride ion in the HR structure (green sphere) is at exactly the same position as the carboxylic group of the D85 side chain in the BR structure. (The figure was prepared using Swiss-PdbViewer public domain software.)

Figure 2

Steady-state absorption spectra of the investigated samples. (a) HR dissolved in 50 mM MOPS buffer at pH 7.5 with no salt (0 M NaCl) or 4 M NaCl added. (b) BR wild-type (broken line) and mutant BR-D85T (line) with different pH values and salt concentrations (as indicated in the graph).

Figure 3

Transient absorption spectra for HR with (a) no (0 M) and (b) high (4 M) NaCl concentration excited at 575 nm. The delay time between −1 ps and 1 ps is plotted on a linear axis and, for longer delay times, on a logarithmic axis. The ultrafast dynamics of both HR samples is not influenced by the salt concentration.

Figure 4

Decay-associated difference spectra of HR (0 M NaCl) for the time constants 0.1 ps, 1.4 ps, and 8.1 ps. The offset spectrum accounts for long-lived intermediates in the photocycle. Because of scattering of the pump pulse, the gray shaded part of the transient spectrum around 575 nm is not shown.

Figure 5

Transient absorption signal for the BR mutants D85T_{high salt, pH 6} (line) and D85T_{low salt, pH 6} (broken line) at a probe wavelength of 510 nm (upper panel) and 678 nm (lower panel). The difference spectrum (dotted line) is calculated according to (A(D85T_{high salt, pH 6}) − 0.75 × A(D85T_{low salt, pH 6})). For comparison, the transient signal of wild-type BR (gray line) is shown (scaled by a factor of 0.25).

Figure 6

Decay-associated spectra of wild-type BR (open circles), D85T_{low salt, pH 6} (squares), and D85T_{high salt, pH 6} (triangles) for the time constants ∼0.5 ps, ∼3 ps, and ∼12 ps. The decay-associated spectra of wild-type BR are scaled by a factor of 0.25.

Figure 7

Steady-state absorption spectra of BR, D85T_{low salt, pH 6}, and D85T_{high salt, pH 6}. The combination of the spectra of BR (30%) and D85T_{low salt, pH 6} (70%) reproduces the spectrum of D85T_{high salt, pH 6}.

Figure 8

Protein structure in the vicinity of the retinal chromophore for wild-type BR (upper part) and the mutant D85S (lower part). The chromophore retinal (RET) and the amino acids Asp²¹² and Asp⁸⁵ (for BR) or Ser⁸⁵ (for the mutant D85S) are shown. The bromide anion of mutant D85S can be located over a wide area (light green). Only with the anion (chloride) in the confined area (dark green) are BR-like primary dynamics observed.