Photophysics of Metalloazurinst

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(I1) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(1I) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(I1) 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 denitrifcans, 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(I1) azurin Pae and Cu(I1) and Hg(I1) azurin Afe. The fluorescence of azurin Pae is assigned as a 'Lb electronic transition, while that of azurin Afe is 'La, The marked fluorescence quenching of Cu(I1) azurin Pae and Cu(1) 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. A z u r i n is a blue copper(I1) protein found in denitrifying soil bacteria and is believed to be involved in electron transport to a terminal oxidase (Henry & Bessiere, 1984; Ryden, 1984; Ambler & Tobari, 1989). The Cu(I1) ion is surrounded by four ligands in a distorted trigonal-planar pyramid arrangement (Baker, 1988)-see Figure 1. The three closest ligands are two histidines and a cysteine, almost equidistant from the Cu(1I) ion, forming a trigonal structure. The fourth ligand is a methionine with a long bond length of 3.1 A (Norris et al., 1986; Baker, 1988). When excited with ultraviolet light, apoazurins exhibit large fluorescence quantum yields, which have been attributed to fluorescence from their tryptophan residues. Azurin from Alcaligenes denitrificans (azurin Ade) contains a buried tryptophan at position 48 and an exposed tryptophan at position 118 (Ambler, 1973; Baker, 1988). Azurins from Pseudomonas aeruginosa (azurin Pae) and Pseudomonasfluorescens (azurin Pfl) contain a single tryptophan at the buried position 48 (Ambler, 1973; Ambler & Brown, 1967; Adman & Jensen, 1981), while azurin from Alcaligenes faecalis (azurin Afe) also contains a single tryptophan at the exposed position 118 (Ambler, 1973). This work was supported by NSF. *Address correspondence to this author. $The University of Chicago. Illinois Institute of Technology. 0006-2960/90/0429-7329$02.50/0 The tryptophan fluorescence in a native azurin is extensively quenched compared to that of its apoprotein (Finazzi-Agro et al., 1973; Szabo et al., 1983; Petrich et al., 1987; Hutnik & Szabo, 1989a). When Cu(I1) is replaced with Cu(I), Ag(I), Hg(II), Co(II), or Ni(II), different amounts of fluorescence quenching are observed (Finazzi-Agro et al., 1973; McMillin et al., 1974; Tennent & McMillin, 1979; Grinvald et al., 1975; Hutnik & Szabo, 1989b). Fluorescence is not quenched when Cu(I1) is replaced with either Cd(I1) or Zn(I1) (Engeseth & McMillin, 1986). The quenching of tryptophan fluorescence could result from several effects, perhaps in combination in some cases. In previous work (Petrich et al., 1987) we proposed that the fluorescence quenching of Cu(I1) azurins occurs via an electron transfer. Long-range electronic energy transfer must also be considered, since in some metalloazurins there is significant absorbance in the region of tryptophan fluorescence. An additional quenching mechanism is an enhanced intersystem crossing as a result of the heavy-atom effect (Finazzi-Agro et al., 1973). The latter process seems unlikely in most systems because luminescence spectra, measured at 77 K, show that the fluorescence to phosphorescence ratio is the same for apoazurin Pfl and Cu(II), Ag(I), and Hg(I1)substituted azurin Pfl (Finazzi-Agro et al., 1973). In addition, heavy-atom quenching is not consistent with structural studies that place the tryptophan 11 A away from the metal atom. Nevertheless, comparison of luminescence spectra measured