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

Abstract

Abstract The 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 (LYS 216 ), the following nearby amino acids (ARG 82 , ASP 85 , ASP 115 , ASP 212 , THR 90 , TRP 86 , TRP 138 , TRP 182 , TYR 57 , TYR 83 , and TYR 185 ) 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 2+ ion near to the chromophore. Four amino acids and three water molecules contribute significantly to the assigned chromophore adjacent calcium binding site (ASP 85 , ASP 212 , TYR 57 and TYR 185 ), and two conformational minima are predicted. The higher energy conformation has the calcium ion stabilized primarily by ASP 85 and the chromophore imine proton by ASP 212 . The lower energy conformation has the calcium ion stabilized primarily by ASP 212 and the imine proton by ASP 85 . The latter configuration is more stable due to strong hydrogen bonding between TYR 185 and ASP 212 coupled with electrostatic stabilization of the divalent cation by TYR 57 . Although both tyrosine residues are predicted to exhibit some “unprotonated” character, models involving full deprotonation of either TYR 57 or TYR 185 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 “ 1 B ” and “ 1 A ” states, despite extensive mixing, exhibit significantly different configurational character. The lowest‐lying “ 1 B ” state is dominated by single excitations (> 80% for all models studied) whereas the second‐excited “ 1 A ” state is dominated by double excitations (> 70% for all models studied with extensive participation by spin‐coupled triplet‐triplet excitations). © 1995 John Wiley & Sons, Inc.