Mutations In Transhydrogenase Change The Fluorescence Emission State Of Trp72 From 1La To 1Lb.

The dI component of Rhodospirillum rubrum transhydrogenase has a single Trp residue (Trp72), which has distinctive optical properties, including short-wavelength fluorescence emission with clear vibrational fine structure, and long-lived, well-resolved phosphorescence emission. We have made a set of mutant dI proteins in which residues contacting Trp72 are conservatively substituted. The room-temperature fluorescence-emission spectra of our three Met97 mutants are blue shifted by ∼4nm, giving them a shorter-wavelength emission than any other protein described in the literature, including azurin from Pseudomonas aeruginosa. Fluorescence spectra in low-temperature glasses show equivalent well-resolved vibrational bands in wild-type and the mutant dI proteins, and in azurin. Substitution of Met97 in dI changes the relative intensities of some of these vibrational bands. The analysis supports the view that fluorescence from the Met97 mutants arises predominantly from the 1Lb excited singlet state of Trp72, whereas 1La is the predominant emitting state in wild-type dI. It is suggested that the sulfur atom of Met97 promotes greater stabilization of 1La than either 1Lb or the ground state. The phosphorescence spectra of Met97 mutants are also blue-shifted, indicating that the sulfur atom decreases the transition energy between the 3La state of the Trp and the ground state.