Kind of blue: Ultra-short wavelength fluorescence from tryptophan in transhydrogenase dI

Until now the Trp in azurin was found to emit fluorescence at a shorter wavelength than Trp residues of other proteins. Trp72 in the dI component of transhydrogenase has a slightly longer emission wavelength but we have isolated three mutants (M97A, M97L and M97V) which emit to the blue of even azurin. Indole has two low-lying excited singlet states (1La and 1Lb) with similar energies. In a vacuum 1Lb lies below that of 1La and is consequently the emission state. The permanent dipole moment of 1La is greater than that of 1Lb and the former is therefore stabilised more by interactions with a protein matrix. It was frequently suggested that azurin is the only known protein to emit from 1Lb. However, fluorescence excitation anisotropy experiments show that azurin, and also wild-type dI, emit predominantly from 1La. In contrast, the three M97 mutants of dI do emit predominantly from 1Lb. This view is supported by comparisons of the intensities of vibrational bands in high-resolution low-temperature fluorescence spectra of wild-type and mutant dI, and azurin, with gas-phase model spectra of indole. Phosphorescence spectra and decays of dI also attest to the exceptional rigidity of its protein core.