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

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 (LYS216), the following nearby amino acids (ARG82, ASP85, ASP115, ASP212, THR90, TRP86, TRP138, TRP182, TYR57, TYR83, and TYR185) 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 Ca2+ ion near to the chromophore. Four amino acids and three water molecules contribute significantly to the assigned chromophore adjacent calcium binding site (ASP85, ASP212, TYR57 and TYR185), and two conformational minima are predicted. The higher energy conformation has the calcium ion stabilized primarily by ASP85 and the chromophore imine proton by ASP212. The lower energy conformation has the calcium ion stabilized primarily by ASP212 and the imine proton by ASP85. The latter configuration is more stable due to strong hydrogen bonding between TYR185 and ASP212 coupled with electrostatic stabilization of the divalent cation by TYR57. Although both tyrosine residues are predicted to exhibit some “unprotonated” character, models involving full deprotonation of either TYR57 or TYR185 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 “1B u*+” and “1A g*−” states, despite extensive mixing, exhibit significantly different configurational character. The lowest‐lying “1B u*+” state is dominated by single excitations (> 80% for all models studied) whereas the second‐excited “1A g*−” 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.