Fluorescence spectra of CHO-wt were recorded in deuterated buffers from pD 6.0 to 10.1 (Table 6.2). Isotopic substitution of all solvent exchangeable protons was used to probe whether the pH-dependence on fluorescence emission was due to a proton transfer in the excited state. The most likely candidate for the proton donor would be the N(3) atom of the flavin because the pKa of the hydrogen bond has been measured to drop from ~10 in the ground state to ~7 in the excited state.45, 57-59 Data reported in Table 6.2 of the wild-type in D2O reveal a similar trend as observed of wild-type enzyme in H2O. The λex and λGS decreased with increasing pD concomitantly as λem was increasing. As shown in Figure 6.3, Panel A, the λem values were plotted as a function of pD and were observed to increase with increasing pD measuring a pKa of 8.4 ± 0.2 with two offsets at low and high pD with limiting values of λlim1 = 525 ± 1 nm and λlim2 = 531 ± 1 nm, respectively. The limiting values calculated in the pD profile of CHO-wt emission in D2O were similar to the enzyme observed in H2O experiments with the pKa shifted upward by ~0.4 units. A pD-dependence on the fluorescence emission was attributed to a proton being transferred during the excited state consistent with an ESPT (Figure 6.2, Panels C-D). A change in the shape of both excitation and emission spectra in the presence of a heavy isotope, D2O, was also consistent with an ESPT (Figure 6.2, Panel B, Panel D).60-62
Table 6.2. UV-Visible absorption and fluorescence spectra maxima of CHO wild-type enzyme obtained at 15 oC in deuterated buffer.a
aReaction conditions are 20 mM NaPPi, pD 6.0, 9.0-10.1 and 20 mM NaPi, pD 7.0-8.0 in deuterated water with spectra measured in a 10 mm quartz cuvette.
Fluorescence spectra of CHO-wt were recorded in buffers containing 10 % (v/v) glycerol as a control for the D2O experiments. Data are shown in Table 6.3. The λmax values of ground and excited state and emission in glycerol-added buffers did not differ more than 5% from λmax values observed in protiated buffers without glycerol. Therefore, the changes in the fluorescence spectra of the wild-type enzyme in deuterated buffer were consistent with an effect due to isotopically-labeled solvent and not a change in the viscosity of the solvent.
Table 6.3. UV-Visible absorption and fluorescence spectra maxima of CHO wild-type enzyme obtained at 15 oC in buffers containing 10 % glycerol (v/v).a
aReaction conditions are 20 mM NaPPi, pH 6.0, 9.0-10.1, 10% glycerol (v/v) and 20 mM NaPi, pH 7.0-8.0, 10% glycerol (v/v) with spectra measured in a 10 mm quartz cuvette.
pD λGS, nm λex, nm λem, nm Stokes Shift, nm
6.0 455 465 525 60 7.0 455 442 526 84 7.5 454 422 525 103 8.0 454 400 527 127 9.0 451 401 530 129 10.0 450 399 531 132
pH λGS, nm λex, nm λem, nm Stokes Shift, nm
6.0 458 469 522 53 7.0 456 469 522 53 7.5 454 443 523 80 8.0 455 419 523 104 9.0 451 400 529 129 10.0 448 400 530 130
6.3.3 pL-Dependence on the Fluorometry of CHO variant, H466Q
UV-Visible absorbance and fluorescence spectral studies were carried out with the H466Q variant enzyme to probe the role of histidine as a catalyst in the ESPT in the active site of CHO. The histidine residue at the 466 position is located on the si face of the flavin cofactor near the N(1)-C(2) locus of the isoalloxazine ring approximately 4.6 Å from the N(3) atom (Figure 6.4). UV-visible absorption, excitation and emission spectra were measured from pH 6.0 to 10.1 at 15 oC with only the spectra at pH 10.1 shown in Figure 6.5. Excited state spectra exhibited red shifts in the ground state from 450 nm to 469 nm from pH 6.0 to 10.1. All spectral peaks (i.e. λGS, λex, λem) at each pH value tested are reported in Table 6.4. The average values for λGS, λex, λem are 453, 468.6, and 520 nm, respectively. Fluorescence spectra of H466Q were also measured in deuterated buffers as prepared for the wild-type enzyme. In deuterated buffer, λmax values of the ground and excited states and emission were 453, 464, and 521 nm, respectively (Table 6.5). The λex and λem values were, thus, independent of pH and pD. Lack of pL effects on λex and λem values were consistent with the absence of ESPT in the H466Q variant. If an ESPT was occurring in the variant enzyme, any amount of C4a flavin intermediate have formed in the excited state and decayed on a time scale faster than that observed in the wild-type enzyme.41 The time scale for the decay of the C4a flavin intermediate would have to be slower for any flavin adduct intermediate to accumulate and therefore be detected.
Figure 6.3 Active site of CHO (4MJW) highlighting H466 and S101 residues.
Table 6.4. UV-Visible absorption and fluorescence spectra maxima of CHO variant enzyme, H466Q, obtained at 15 oC.a
aReaction conditions are 20 mM NaPPi, pH 6.0, 9.0-10.1 and 20 mM NaPi, pH 7.0-8.0 with spectra measured in a 10 mm quartz cuvette.
Table 6.5. UV-Visible absorption and fluorescence spectra maxima of CHO variant enzyme, H466Q, obtained in deuterated buffer at 15 oC.a
aReaction conditions are 20 mM NaPPi, pD 6.0, 9.0-10.1 and 20 mM NaPi, pD 7.0-8.0 with spectra measured in a 10 mm quartz cuvette.
pH λGS, nm λex, nm λem, nm Stokes Shift, nm
6.0 455 469 521 52
7.0 451 471 519 48
8.0 453 469 519 50
9.0 455 466 521 55
10.1 451 468 521 53
pD λGS, nm λex, nm λem, nm Stokes Shift, nm
6.0 453 464 522 58
7.0 453 463 520 57
8.0 454 464 521 57
9.0 453 464 521 57
Figure 6.4 Fluorescence and absorption spectroscopy of H466Q variant enzyme in 20 mM sodium pyrophosphate, pH 10.1.
Spectra are for absorbance (black), fluorescence excitation (blue), and fluorescence emission (red) at 15 oC, in 20 mM sodium pyrophosphate, pH 10.1.