title: Photophysics of metalloazurins.
abstract: The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.
authors.affiliation: Department of Chemistry, University of Chicago, Illinois 60637.
authors.forename: J E
authors.initials: JE
authors.lastname: Hansen
authors.forename: J W
authors.initials: JW
authors.lastname: Longworth
authors.forename: G R
authors.initials: GR
authors.lastname: Fleming
mesh_terms: D000421:Alcaligenes / Q000032:analysis; D001400:Azurin / Q000737:chemistry* / Q000528:radiation effects; D001426:Bacterial Proteins / Q000737:chemistry*; D008667:Metalloproteins / Q000737:chemistry* / Q000528:radiation effects; D010777:Photochemistry; D011550:Pseudomonas aeruginosa / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014364:Tryptophan
chemical_list: D001426:Bacterial Proteins; D008667:Metalloproteins; D001400:Azurin; D014364:Tryptophan