The active site of bacteriorhodopsin. Two‐photon spectroscopic evidence for a positively charged chromophore binding site mediated by calcium

Abstract

SYNOPSISThe nature of the chromophore binding site of light-adapted bacteriorhodopsin is analyzed by using all-valence electron MNDO and MNDO-PSDCI molecular orbital theory to interpret previously reported linear and nonlinear optical spectroscopic measurements. A total of 45 binding site models are investigated. The binding site is simulated by including the chromophore, the lysine residue (LYSZl6), the following nearby amino acids ASP,,, ASPIIS, ASPZIZ, THRgo, TRPa6, T R P I~s , TRPlsz, TYR57, TYRs3, and TYRIs5) and zero, one, or two divalent cations. We conclude that the unique two-photon properties of the chromophore are due in part to the electrostatic field associated with a Ca'+ ion near to the chromophore. Four amino acids and three water molecules contribute significantly to the assigned chromophore adjacent calcium binding site (ASPs5, ASP,,,, TYRS7 and TYR,,,), and two conformational minima are predicted. The higher energy conformation has the calcium ion stabilized primarily by ASP,, and the chromophore imine proton by ASP,,,. The lower energy conformation has the calcium ion stabilized primarily by ASPzl2 and the imine proton by ASPs5. The latter configuration is more stable due to strong hydrogen bonding between TYRIs5 and ASP212 coupled with electrostatic stabilization of the divalent cation by TYRS7. Although both tyrosine residues are predicted to exhibit some "unprotonated' character, models involving full deprotonation of either TYR57 or TYRls5 do not fit the spectroscopic data. We conclude that the cation binding site identified in this study is the second high affinity binding site for calcium, and that the chromophore binding site is, to a first approximation, positively charged.The chromophore "lh*t" and "I$*-" states, despite extensive mixing, exhibit significantly different configurational character. The lowest-lying '"&*+" state is dominated by single excitations (> 80% for all models studied) whereas the second-excited "lAg*-'' state is dominated by double excitations (> 70% for all models studied with extensive participation by spin-coupled triplet-triplet excitations).

Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin

“…The PSB group is now close to Glu-181 to establish the electrostatic interaction (green dashed line) with the new counterion. In the Meta intermediates, it is possible that the counterion environment about Glu-181 is complex as has been observed and discussed in the case of bacteriorhodopsin (30,31).…”

“…In addition to the known anion effects (29), these interactions could be a determining factor controlling the deprotonation of the SB in native Meta II. This suggests that, as indicated by the structure, Glu-181 and Glu-113 are not interacting independently with the chromophore in the later intermediates such that the possibility of a complex counterion as observed in bacteriorhodopsin must be considered (30,31).…”

Detection of a Yb 3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

“…A number of spectroscopic (26,27) and potentiometric titration (21,22) studies of bR and some of its mutants lead to the conclusion that Ca 2ϩ binds to amino acid residues not far from the retinal. Furthermore, Ca 2ϩ in the second highaffinity site (7,8,22) is responsible for the color change in regenerated bR.…”

“…This suggests the presence of Ca 2ϩ near D85, which is the active site of bR. Most recently, Stuart et al (27) could calculate the two photon spectrum of the retinal in bR only if the Ca 2ϩ is placed near D85 and D212 within the active site.…”

Contribution of Extracellular Glu Residues to the Structure and Function of Bacteriorhodopsin

“…Some authors claim a nonspecific location of the cations in the double layer (18,21,22) or in the lipid phase (26). However, the majority of works argue for the existence of specific binding sites in the purple membrane, including a site near Asp 85 (24,27,28), or a more external location (29,30). Among the most outstanding results pointing toward specific cation locations in the protein moiety are: (i) the evidence for carboxyl participation in cation binding from studies using specific reagents (13,17); (ii) the presence of fewer cation-binding sites and with lower affinity in the bleached and pink membranes as compared with purple membranes (14,16,19), even that the electric surface potential of bleached membrane remains unchanged (31); (iii) extended x-ray absorption fine structure data describing a different environment for the bound Mn 2ϩ with respect to free Mn 2ϩ in water and ruling out the interaction of Mn 2ϩ with P or S atoms (32); (iv) Fourier transform infrared studies showing that binding of Mn 2ϩ to deionized membrane induces changes in the reverse turns, located in the loops (33); and (v) 13 C NMR studies detecting changes in the Ala 196 environment upon divalent cation binding (25).…”