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

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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.