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 (Trp(72)), 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 Trp(72) are conservatively substituted. The room-temperature fluorescence-emission spectra of our three Met(97) mutants are blue shifted by approximately 4 nm, 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 Met(97) in dI changes the relative intensities of some of these vibrational bands. The analysis supports the view that fluorescence from the Met(97) mutants arises predominantly from the (1)L(b) excited singlet state of Trp(72), whereas (1)L(a) is the predominant emitting state in wild-type dI. It is suggested that the sulfur atom of Met(97) promotes greater stabilization of (1)L(a) than either (1)L(b) or the ground state. The phosphorescence spectra of Met(97) mutants are also blue-shifted, indicating that the sulfur atom decreases the transition energy between the (3)L(a) state of the Trp and the ground state.
Tveen Jensen
Karina
K
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
Strambini
Giovanni
G
Gonnelli
Margherita
M
Broos
Jaap
J
Jackson
J Baz
JB
D019943:Amino Acid Substitution; D005453:Fluorescence; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan
D050505:Mutant Proteins; D014364:Tryptophan; D009250:NADP Transhydrogenases