Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs

Abstract

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.

Crystal Structures of Archaerhodopsin-1 and -2: Common Structural Motif in Archaeal Light-driven Proton Pumps

“…This result is in line with the previous observation of a higher content (77%) of the trans isomer in the dark adapted state of aR-2. 24 In Figure 6, the structures of the cytoplasmic halves of the three proton pumps (bR, aR-1 and aR-2) are compared. It shows that the structural conservation is very low for residues extruded from helices E and F. This would be expected from the low sequence homology for residues in these helices (Figure 1).…”

“…This difference is believed to come from substitution of a residue (Met145 in bR) contacting the retinal ionone ring, since the same thermodynamic property as observed for aR-2 is reproduced when Met145 in bR is substituted to phenylalanine. 24 It was reported, on the other hand, that the pK a of the acid purple-to-blue transition is much higher in aR-2 than in aR-1 or bR. 22 It has not been clarified whether this difference correlates with a difference in the amino acid sequence.…”

Structure of bacteriorhodopsin at 1.55 Å resolution

“…Site-speci®c mutagenesis of many of these residues resulted in changed absorption maxima or changed rate of thermal isomerization (e.g. see Marti et al, 1991;Sonar et al, 1993;Ihara et al, 1994;Delaney et al, 1995;Weidlich et al, 1996). The side-chain of Leu93, a residue that was found to in¯uence the rate of 13-cis-to all-trans reisomerization in the photocycle (Delaney et al, 1995), is as close as 3.6 A Ê from the 13-methyl group of the retinal.…”

Halobacterial Rhodopsins

“…The bR with a point-mutation at this assigned residue, M145FbR, was expressed in halobacteria and found to give an isomer ratio of 3:1 the same as in aR-2 (49). In aR-2, this equilibrium reaches an isomer ratio of only about 3:1 (48).…”