Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection?

One of the first events taking place when a crystal of a metalloprotein is exposed to X-ray radiation is photoreduction of the metal centres. The oxidation state of a metal cannot always be determined from routine X-ray diffraction experiments alone, but it may have a crucial impact on the metal's environment and on the analysis of the structural data when considering the functional mechanism of a metalloenzyme. Here, UV-Vis microspectrophotometry is used to test the efficacy of selected scavengers in reducing the undesirable photoreduction of the iron and copper centres in myoglobin and azurin, respectively, and X-ray crystallography to assess their capacity of mitigating global and specific radiation damage effects. UV-Vis absorption spectra of native crystals, as well as those soaked in 18 different radioprotectants, show dramatic metal reduction occurring in the first 60 s of irradiation with an X-ray beam from a third-generation synchrotron source. Among the tested radioprotectants only potassium hexacyanoferrate(III) seems to be capable of partially mitigating the rate of metal photoreduction at the concentrations used, but not to a sufficient extent that would allow a complete data set to be recorded from a fully oxidized crystal. On the other hand, analysis of the X-ray crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than metal centre reduction, and suggests further testing of HEPES and 2,3-dichloro-1,4-naphtoquinone as potential scavengers.

Ligand binding and protein dynamics in cupredoxins.

Type 1 copper sites bind nitric oxide (NO) in a photolabile complex. We have studied the NO binding properties of the type 1 copper sites in two cupredoxins, azurin and halocyanin, by measuring the temperature dependence of the ligand binding equilibria and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6)-10(2) s). In both proteins, we find nonexponential kinetics below 200 K that do not depend on the NO concentration. Consequently, this process is interpreted as geminate recombination. In azurin, the rebinding can be modeled with the Arrhenius law using a single pre-exponential factor of 10(8.3) s-1 and a Gaussian distribution of enthalpy barriers centered at 22 kJ/mol with a width [full width at half-maximum (FWHM)] of 11 kJ/mol. In halocyanin, a more complex behavior is observed. About 97% of the rebinding population can also be characterized by a Gaussian distribution of enthalpy barriers at 12 kJ/mol with a width of 6.0 kJ/mol (FWHM). The pre-exponential of this population is 1.6 x 10(12) s-1 at 100 K. After the majority population has rebound, a power-law phase that can be modeled with a gamma-distribution of enthalpy barriers is observed. Between 120 and 180 K, an additional feature that can be interpreted as a relaxation of the barrier distribution toward higher barriers shows up in the kinetics. Above 200 K, a slower, exponential rebinding appears in both cupredoxins. Since the kinetics depend on the NO concentration, this process is identified as bimolecular rebinding.(ABSTRACT TRUNCATED AT 250 WORDS)

Room-temperature phosphorescence from azurin derivatives. Phosphorescence quenching in oxidized native azurin.

The tryptophan phosphorescence from a series of derivatives of Pseudomonas aeruginosa azurin has been monitored at 30 degrees C in pH 8.5 buffer solution. The phosphorescence lifetimes fall in the range of 230-270 ms for deoxygenated solutions of derivatives containing Cd(II), Cu(I), Co(II), Ni(II), Hg(II) or apoazurin. A weak signal with a lifetime of ca 130 ms is observed from solutions of oxidized native azurin, but this component is ascribed to a modified form of azurin in solution, i.e. protein heterogeneity, on the basis of the unique sensitivity to quenching by dioxygen. Aside from this minor component, the tryptophan phosphorescence in the Cu(II) protein appears to be fully quenched. The quenching is assigned an electron-transfer mechanism involving transient reduction of the metal center. The same mechanism is deemed to be responsible for fluorescence quenching in oxidized native azurin as well. These observations are of interest because aromatic groups like tryptophan may be conduits for physiological electron-transfer processes involving the copper center.

Photophysics of metalloazurins.

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.