Investigation of the binding mechanism and inhibition of bovine liver catalase by quercetin: Multi-spectroscopic and computational study

Kinetics studies of BLC

The activity of BLC was determined using a UV spectrophotometer (T60, PG Instruments LTD., Leicestershire, UK) by detecting the decrease in H₂O₂ maximum absorbance at 240 nm (A240) in sodium phosphate buffer (pH= 7, 50 mM), due to its break down by the enzyme. To investigate the effects of QUE on the activity of BLC, its 3 nM solution was incubated with different concentrations of QUE (5.5, 11, 16.5, 22 and 27.5 μM), in a total volume of 3 mL. Subsequently, a fixed concentration of H₂O₂ (70 mM) was added to the solutions and the absorbance of them was recorded at 240 nm every 2 seconds for 1 minute.

Spectroscopic investigations

BLC absorption spectra were recorded in the range of 200-500 nm, using 1 cm quartz cell. The fluorescence spectra were recorded using a FP-750 Jasco spectrofluorometer (Kyoto, Japan) at 2 temperatures (25°C and 37°C) with the excitation wavelength (λ_ex) of 280 nm and the fluorescence emission wavelength (λ_em) range of 300-500 nm. The synchronous fluorescence spectra of BLC with and without QUE were scanned using Δλ=15 nm (λ_em=240-400) for tyrosine (Tyr) and Δλ=60 nm (λ_em=300-500) for tryptophan (Trp). The scan speed was fixed at 1000 nm.min-¹.

CD spectra of BLC were recorded on a J-810 spectropolarimeter (Jasco, Tokyo, Japan) in the presence and absence of QUE at room temperature in 0.1 cm cell in 50 mM sodium phosphate buffer. The percentage of secondary structure elements of BLC upon interaction with various concentrations of QUE, were estimated by the CDNN software.

Molecular docking study

Molecular docking was carried out to obtain the best binding site of QUE on BLC using the Auto Dock software (version 4.2).¹⁷ BLC's crystal structure (PDB ID: 1TGU) was taken from the Protein Data Bank. QUE molecular structure was drawn by Hyperchem 8.0.6 and its energy minimized conformation was conducted using Gaussian 03 programs. The Lamarckian genetic algorithm (LGA) search method was used for searching the best binding site of QUE to the BLC. The modes of interaction between BLC and QUE was investigated using Chimera 1.10.1 program.¹⁸

Analysis of BLC activity

Catalase breakdowns the H₂O₂ to H₂O and O₂. The rate of decomposition of H₂O₂ by BLC, which leads to decrease in H2O2 absorption at 240 nm, was considered as the enzyme activity. For this purpose, the additional concentrations of H₂O₂ (10-90 mM) in the presence of the fixed concentration of BLC (3 nM) was used to determine the BLC activity. The results indicated that with raising H₂O₂ concentration, BLC activity increases (from 10 to 70 mM), after that with more concentrated solutions of H₂O₂ (> 70 mM), a significant reduction in BLC activity was detected, due to suicide inactivation process.¹⁹ Lineweaver-Burk plot and equation 3 were applied to determine the accurate kinetic parameters.

\[\frac{1}{V}{\text{ = }}\frac{{{K_m}}}{{{V_{\max }}}}.\frac{1}{{[S]}} + \frac{1}{{{V_{\max }}}}\]

The K_m (Michaelis Constant) and V_max (Maximal Velocity) values were obtained to be 39 mM and 2.28 mM.S^-1, respectively. Also, the equation (4) was used to estimate the catalytic constant (K_cat) value, which was obtained as 7.6 × 10⁵ s-¹.

\[{V_{\max }} = {K_{cat}}{\text{.[}}{{\text{E}}_t}{\text{]}}\]

where, is total enzyme concentration.

In order to investigate the effects of QUE on enzyme activity, BLC activity assays were performed in the presence of additional concentrations of QUE (5.5, 11, 16.5, 22 and 27.5 μM) and fixed concentration of H₂O₂ (70 μM). It has been observed that with an increase in QUE concentration, BLC activity markedly reduced. According to Fig. 2A, the IC₅₀ value was calculated as 16.76 μM. The inhibition kinetics of BLC by QUE was then analyzed by a Lineweaver-Burk plot (Fig. 2B) which indicated that QUE is a non-competitive inhibitor of BLC. When the slope of each curve from Fig. 2B was re-plotted against different concentrations of QUE a K_i of 46.0 μM was obtained (Fig. 2C).

Fig. 2. Effect of different concentrations of quercetin (0, 5.5, 11, 16.5, 22 and 27.5 µM) on the catalytic activity of bovine liver catalase in phosphate buffer 50 mM, pH 7, at room temperature. (B) Lineweaver-Burk curve of bovine liver catalase (3 nm) in the absence and presence of different concentrations of quercetin: 0 (×), 8.38 (♦), 16.76 (■) and 33.52 (▲) µM in phosphate buffer 50 mM, pH 7, at room temperature. (C) The secondary linear plot which derived of Lineweaver–Burk plot.

UV-vis spectrometric investigations

UV-vis absorption spectrometry is a simple and effective method, which is applied to investigate the structural change of proteins upon interaction with ligands. The UV-vis spectrum of BLC shows the main absorption band around 280 nm, which mainly comes from aromatic amino acids. Also, BLC possesses another significant peak around 405 nm (Soret-band), which is caused by the π→π^* transition of electrons in the heme group.^{20, 21} UV-vis spectra of BLC (1 μM) were measured in different concentrations of QUE (8.38, 16.76 and 33.52 µM). The results indicated that with increasing concentration of QUE both of the absorption peaks (280 and 405 nm) increase, but more investigations showed that QUE has its own absorption in the studied wavelengths (280 and 405 nm), so the absorption values of QUE at different concentrations were subtracted from the absorption values of BLC-QUE, showing that the interaction between BLC and QUE result in a little hyperchromic effect around 280 nm (Fig. 3A) and 405 nm (Fig. 3B). These results indicated that QUE can form complex with BLC and alter its structure slightly.¹⁹

Fig. 3. The changes in bovine liver catalase absorption (1 µM) at 280 nm (A) and 405 nm (B) after addition of quercetin in phosphate buffer 50 mM, pH 7, at room temperature.

Fluorescence spectroscopy

Fluorescence spectroscopy is a fast, robust and highly sensitive tool, which is applied to study the structural changes of macromolecules upon interaction with various ligands. The intrinsic fluorescence emission of BLC results from aromatic fluorophores (dominantly Trp and Tyr residues). The intrinsic fluorescence emission of tryptophan is very sensitive to the polarity of the surrounding environment.²² We used fluorescence spectroscopy to investigate the interaction between QUE and BLC. As can be seen from Fig. 4, the fluorescence intensity of BLC decreases in the presence of an additional concentration of QUE (1, 2, 3, 4, 5 and 6 µM). Results indicated that QUE can quench the intrinsic fluorescence of BLC and change the micro-region environment around the fluorophores (excitation wavelength was set at 280 nm).

Fig. 4. Effect of concentration of quercetin on fluorescence intensity of bovine liver catalase before and after addition of quercetin. λex=280 nm, BLC concentration: (0.7 µM); quercetin concentrations:(a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5, (g) 6.

Fluorescence quenching mechanisms are classified into 2 types by their different dependence on temperature: dynamic (collisional quenching) and static (non-fluorescent complex formation). In order to estimate the quenching mechanism of BLC in the presence of QUE, Stern-Volmer equation (5) was used:

\[\frac{{{F_0}}}{F}{\text{ = 1 + }}{{\text{K}}_{sv}}{\text{[Q] = 1 + }}{{\text{K}}_q}{\tau _0}{\text{[Q]}}\]

where, F and F₀ are the fluorescence intensities of BLC with and without the quencher (QUE), respectively, [QUE] is the concentration of the quencher, K_q is the quenching rate constant of the biomolecule, τ0is the fluorescence lifetime without quencher (10-8 s for BLC) and KSVis the Stern–Volmer quenching constant. The careful examination of the plot of F₀/F vs. [QUE] (Fig. 5) revealed that the K_SV values decrease with an increase in temperature, and K_q values are much greater than 2×10¹⁰ L mol-¹ s-¹, which are characteristic of static quenching mechanism.²³

Fig. 5. Stern-Volmer plot for fluorescence quenching of bovine liver catalase by different concentrations of quercetin at 298 K (♦) and 310 K (■).

Synchronous fluorescence spectroscopy

Synchronous fluorescence spectroscopy provides important information about the micro-region around the fluorophores. By scanning simultaneously both excitation and emission wavelengths, maintaining a constant wavelength interval (∆λ) between them (∆λ=15 nm for Tyr and ∆λ=60 nm for Trp).²⁴ The gradual decrease in the fluorescence intensity on the successive additions of QUE is an obvious indication of the complex formation between BLC and QUE, but the peak positions of the synchronous fluorescence spectra remain unshifted (Fig. 6A and 6B). It seems that the binding of QUE to BLC has no obvious effect on the microenvironment around the Tyr and Trp residues.²⁵

Fig. 6. Synchronous fluorescence emission spectra of bovine liver catalase in the presence of different concentrations of quercetin (0, 1, 2, 3 and 4 µM); (A) ∆λ=15 and (B) ∆λ=60 nm, (f) only quercetin 1 µM, at room temperature.

Circular dichroism spectroscopy study

To gain a better understanding of QUE effects on the secondary structure of BLC, CD spectroscopy was used. Fig. 7 shows the CD spectra of BLC in the presence or absence of different concentrations of QUE in the far-UV spectral region (190-250 nm). The CD spectra of BLC exhibits two negative bands at 208 and 222 nm (characteristic of the α-helical structure of the protein) and a negative band around 217 nm (related to β-sheets). The CD spectra recorded were analyzed using CDNN software and obtained results were summarized in Table 1. According to Table 1, the content of the α-helical structure in BLC increased from 26.4% to 30.3%, while the content of β-sheet decreased from 21.7% to 19.1%, suggesting the considerable changes in the protein secondary structure. Also, the results indicate that QUE can increase the stability of BLC via increasing the α-helical structures and reduction of other secondary elements of protein.²⁶

Fig. 7. Change in CD spectra of bovine liver catalase with and without various concentrations of quercetin at room temperature.

Thermodynamic analysis and binding mode

In order to calculate the number of binding sites (n) and binding constant (K_a), the following equation was used:

\[{\text{log}}\frac{{{F_0} - F}}{F} = \log {K_a} + n\log [Q]\]

According to a plot of vs. log [QUE] (Fig. 8), the K_a and "n" values were obtained and the results have been summarized in Table 2. The results indicated that there is only one binding site on BLC for QUE (n ~ 1) and the K_a value increased with the increasing temperature, suggesting that the stability of complex increase with the rising temperature. The K_a value was about 10⁴, indicating that there is a strong interaction between QUE and BLC.²⁷

Fig. 8. The linear plot of / vs. log [quercetin], according to equation 7, for fluorescence quenching of bovine liver catalase in the presence of quercetin at 298K (♦) and 310K (■).

There are various kinds of acting forces between a ligand and a macromolecule, which are classified into four groups based on the sign and the magnitude of the calculated parameters: 1) ∆H˂ 0 and ∆S > 0 (electrostatic forces play the main role), 2) ∆H ˃ 0 and ∆S ˃ 0 (hydrophobic interactions are important), 3) ∆H ˂ 0 and 4) ∆S ˂ 0 (van der Waals forces and hydrogen bonding are dominant).²⁸Table 2 summarized the numerical values of ∆H, ∆S and ∆G calculated according to equations 8-10.

\[\ln \frac{{{ K_2 }}}{{{K_1}}} = \frac{{ - \Delta H}}{R}(\frac{1}{{{T_2}}} - \frac{1}{{{T_1}}})\]

\[\Delta G = - RT\ln K\]

\[\Delta G = \Delta H - T\Delta S\]

The positive values of enthalpy changes (∆H) and the entropy changes (∆S) indicate that the hydrophobic interactions are involved in the complex formation between BLC and QUE. Also, the ΔG values are negative, indicating that interaction of QUE with BLC is a spontaneous process.^{23, 27}

Molecular docking results

To predict the best binding sites of QUE on BLC molecular docking studies were performed. Fig. 9 (A and B) shows that the best binding site of QUE on BLC is located away from heme group, at a cavity among the wrapping domain, threating arm and β-barrel, confirmation that the QUE is a non-competitive inhibitor. Fig. 9C shows the involving amino-acid residues in the interaction of BLC and QUE (Pro 367, Gln 386, Gln 397, Met 394, Asn 368, Tyr 369, Gln 371, Leu 370, Cys 376, Pro 377 and Val 382). It is clear that QUE interacts with BLC through hydrophobic interaction and hydrogen bonding, but hydrophobic interactions are dominant. The molecular docking results illustrated that there is one binding site for QUE on BLC. The value of lowest binding free energy was calculated as -7.21 kcal mol-¹.

Fig. 9. Molecular docking results for complex of bovine liver catalase with quercetin (A) The carton form of one subunit of bovine liver catalase and (B) in hydrophobic surface model. (C) bovine liver catalase and quercetin amino acid residues in the binding site.