Fig 1.
UV-vis molecular absorption and EPR spectra of the blue, yellow and artificial adducts of the S. sclerotiorum laccase.
A. UV-vis spectra of blue and yellow forms of the laccase of S. sclerotiorum in 25 mM MES buffer pH 6.3. The protein concentration is 7.5 μM in both cases. B. EPR spectra of blue and yellow laccase forms revealing both the type 1 copper center and the type 2 copper center. Conditions: 25 mM MES pH 6.3, 60 μM laccase. C. UV-vis spectra of the blue (a) and yellow (b) laccases, and of the red guaiacol (c), orange TMB (d), and purple ABTS (e) laccase adducts. The spectra are normalized at 280 nm. The approximate concentration of the protein (possible ε280nm variation) is 8 μM. D. ABTS-tyrosine (blue) and ABTS-laccase (red) UV-vis spectra at pH 6.3 (25 mM MES).
Fig 2.
Free tyrosine—ABTS● interaction and pH titration.
A. Tyrosine and ABTS● form an adduct in a 1:1 ratio, as deduced from a Job’s plot. B. The solutions’ spectra are measured after overnight reaction of ABTS● with tyrosine in different molar reaction ratios ([Tyr]/[ABTS]) as labeled, in presence of 15 nM laccase (to ensure radical form of the ABTS), 50 mM citrate buffer, pH 4. C. pH titration of ABTS-tyrosine (circles) and ABTS-laccase (rhombuses) adducts. The pKa values are calculated by fitting the experimental data with a sigmoidal model (see text). D. UV-vis changes of ABTS-laccase adduct while pH decreases.
Fig 3.
MS analyses of the ABTS-laccase adduct.
A. HPLC chromatograms of the tryptic digests of blue laccase and ABTS-laccase as monitored at 230 nm and 555 nm. The most important peptides peaks are indicated by arrows and their corresponding m/z and charge. The 11.5 min peak corresponding to the peptide that was present in the blue form but modified in the laccase-ABTS adduct has m/z 973.47, z = 2. The 12.25 min peak corresponds to the modified peptide (two merged adducts, m/z 1228.46 and 1107.97, z = 2 is also indicated. B. Separation of the two adducts of the same modified peptide using a high-resolution column. C. The MS of the two adducts. D. The UV-vis spectrum of the two adducts. E. Chemical formulas and proposed structures of the two adducts formation and tentative mechanism of formation.
Fig 4.
Molecular absorption and fluorescence spectroscopic investigation of the yellow and artificial adduct-laccase forms.
A. Reduction of pure yellow laccase (a) with dithionite in excess (b) and re-oxidation by exposition to air (c) monitored by UV-vis spectroscopy. Inset: Blue form (solid black), guaiacol-laccase form (solid gray) and difference spectra for yellow laccase (dotted): a–b (isolated oxidized–reduced), c–b (re-oxidized–reduced), and a–c (isolated oxidized–re-oxidized). B. Fluorescence spectra (emission while exciting at 330 nm and excitation while followed at 420 nm) of blue, yellow and ABTS-laccase. Protein solutions are in 20 mM TrisHCl pH 6.8. C. Titration of yellow, blue and ABTS-laccase with guanidine hydrochloride monitored by the shift of the maximum band of the emission spectrum while the sample was excited at 280 nm, using fluorescence spectroscopy. The experimental data was fitted with sigmoidal curves, yielding inflection points: yellow laccase (circles) 2360 ± 67; ABTS-laccase (triangles) 2616 ± 36; blue laccase (rhombuses) 2835 ± 57. D. Exponential decays of activity of yellow, blue and ABTS-laccase while exposed at 50○C. The half-lives are 3.5, 0.8 and 1.1 h respectively while at 40○C are 8.2, 5.6 and 6.8 h respectively. E. Optimal temperature determination for the blue (squares), yellow (triangles) and ABTS adduct (rhombuses). F. Typical Arrhenius plot in the 30–60○C range for the activation energy determination.
Table 1.
Michaels-Menten parameters for three substrates for five forms of the purified laccase obtained by Eadie–Hofstee linearization in case of biphasic cases (Q0H2) and non-linear fitting using GraphPad for normal curves.
Relative standard deviations are 5–20%.
Fig 5.
Structural implications of the Y65.
A. Localization of the Tyr65 relative to the T2/3 and T1 sites. B. BLAST results of the key tyrosine hydrophobic loop present only in Ascomycota, showing its presence in other multicopper oxidases. The Y seems to be well conserved. C. Putative water channel from the trinuclear site to the protein surface and the gating position of the Y65. D. Distance between H133 and Y65.