Two-photon double resonance spectroscopy of bacteriorhodopsin. Assignment of the electronic and dipolar properties of the low-lying 1A*−g-like and 1B*+u-like π, π* states

Abstract

The electronic and dipolar properties of the all-trans retinyl polyene in light-adapted bacteriorhodopsin are examined by using two-photon double resonance spectroscopy to assign the Franck–Condon maxima, the absolute two-photon absorptivities and the change in dipole moments upon excitation of the low-lying ‘‘forbidden’’ 1A*−g -like and ‘‘allowed’’ 1B*+u -like π, π* excited singlet states. The second-order hyperpolarizability is also determined. The two-photon double resonance spectrum, collected with laser excitation from 820–1200 nm in 10 nm steps, displays two maxima, an intense band at ∼18 000 cm−1 assigned to the 1B*+u -like π, π* excited singlet state and a weaker shoulder at ∼21 000 cm−1 assigned to the 1A*−g -like π, π* excited singlet state. Thus, the 1A*−g -like state is 3500±500 cm−1 above the 1B*+u -like state, which is indicative of a protonated Schiff base chromophore. A log-normal fit of the two-photon spectrum indicates that the maximum two-photon absorptivity of the 1B*+u -like state is 290±50 GM whereas the maximum two-photon absorptivity of the 1A*−g -like state is less than half this value, 120±90 GM. The ‘‘1B*+u ’’ state exhibits an absorptivity that is dominated by initial and final state contributions to the two-photon tensor, and this observation allows an accurate assignment of the change in dipole moment upon excitation yielding Δμso=13.5±0.8 D. A similar analysis of the ‘‘1A*−g’’ state predicts that the change in dipole moment upon excitation into the latter state is slightly smaller (Δμso=9.1±4.8 D). We demonstrate that the second-order hyperpolarizability of a molecule can be determined directly from the two-photon absorptivity of the low-lying charge transfer state and other spectroscopic parameters, all but one of which can be determined directly from experiment. Our analysis of light adapted bacteriorhodopsin indicates that β=βxxx+(1/3)[βxyy +2βyyx+βxzz+2βzzx] =(2250±240)×10−30cm5/esu for a laser wavelength of 1.06μ assuming that the homogeneous linewidth is 250 cm−1. Preliminary analyses of the two-photon data indicate that the chromophore in bacteriorhodopsin is a protonated Schiff base chromophore in a very ionic, and possibly charged, binding site. The two-photon data are not consistent with charged species near the β-ionylidene ring, but are consistent with polar species near the β-ionylidene ring. Direct hydrogen bonding of a negative counterion with the imine proton is not supported by the two-photon data.