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

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 2...