CuZnSOD from bovine erythrocytes was purchased from MP Biologicals. Flavonoids were purchased from Indofine Chemical Corporation. Buffers, NaF, trifluoroacetate (tfa), and other reagents were purchased from com- mercial sources and used without purification. ¹⁹F NMR spectra were acquired at 283 MHz (300 MHz ¹H frequency) using a JEOL Eclipse NMR spectrometer. NMR solutions were prepared with 20 mM buffer, 20 mM NaF, 10% (v/v) D2O, and sufficient enzyme to give an easily measurable increase in transverse relaxation rate (R₂ = 1/T₂). Typically, the effects of potential inhibitors were measured under conditions where inhibitor-free, enzyme-containing control gave T₂ values between 7 and 50 ms.

Screening experiments employed a one-dimensional experiment using 2 mM trifluoroacetate (tfa) as an internal reference. While the one dimensional experiments employed the CPMG pulse sequence, spectra were acquired at a single relaxation delay. The ¹⁹F resonance integrals for fluoride and tfa were compared to determine the effects of potential inhibitors on the NMR relaxation activity of the enzyme.

CuCl₂ or CuZnSOD (50 µM), were allowed to incubate for known times with or without 10 mM polyphenol in 100 mM buffer, (either Tris, pH 8 or glycine/NaOH, pH 11) at room temperature. Samples without buffer were prepared using the same concentrations in deionized water, and the pH was adjusted by addition of NaOH to the appropriate value. Sample volumes were 0.2 mL. EPR spectra were measured at 120 K on a Bruker EMX spectrometer in quartz EPR tubes (0.3 mm i.d.). EPR samples were flash-frozen in liquid nitrogen and loaded into a pre-cooled cavity. Reported spectra are the average of five scans with a modulation amplitude of 10 G, modulation frequency of 100 kHz, microwave power of −1.0 mW, microwave frequency of 9.432 GHz, time constant of 40.96 ms, conversion time of 81.92 ms, and a sweep width of 1000 G centered at 3100 G. Spectra were corrected for residual cavity signal.

A 2 mL sample containing approximately 4400 units of SOD (20 mM glycine buffer, pH 10) was divided into two aliquots and 16.2 μL of either DMSO or 123 mM methyl 3,4-dihydroxybenzoate (4, MDHB) solution in DMSO were added. The control and 4 containing solutions were allowed to equilibrate overnight at room temperature. Aliquots of the experimental and control solutions were removed for analysis of total copper and the high molecular weight components were removed from the remaining samples using YM-10 membrane filtration devices (10 KDa molecular weight cutoff, Millipore). Aliquots of the four samples were treated with aqueous HNO₃ and copper concentrations were determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) using a Perkin Elmer ICP-OES Optima 4100 DV spectrometer.

DMSO solutions with a range of polyphenol concentrations were prepared by serial dilution at 50 times their intended final concentration. NMR samples were prepared by addition of 12 µL aliquots of these DMSO solutions to 588 µL enzyme/buffer/fluoride solution. Thus, NMR solutions containing varied concentrations of polyphenol were prepared with a constant 2% (v/v) DMSO. Fluoride ion ¹⁹F transverse relaxation rates were determined by the standard CPMG method [32] . Fractional enzyme activities (A) in the NMR assay were calculated for inhibited solutions according to Equation (1);

where R_2,obs, R_2,+ and R_2,− are the transverse relaxation rates of the sample and controls containing either the same amount of enzyme or none, respectively. Inhibition profiles were determined by measuring fractional activities of samples as a function of polyphenol concentration.

The Molecular Operation Environment (MOE) program (Chemical Computing Group, Montreal) was used to dock nine flavonol structures to the active site of molecule 1 from the 1PU0 structure of CuZnSOD. Flavonolstructures were built and energy minimized in MOE as anions, deprotonated at the 4’ position. The enzyme structure was energy minimized after proton positions and partial charges had been assigned. Atoms within 12 Å of the copper atom were considered part of the active site. The flavonols were docked to the selected active site using the Triangle Matcher placement method. The docked poses were energy minimized using Force Field minimization and the energies of the poses were scored using the London DG method.

Samples of enzyme in buffered solution were sparged with argon and aliquots of either MTHB or quercetin were added anaerobically. Using the NMR assay, samples were tested periodically for several hours, and again after overnight incubation.

The effects of oxidized CuZnSOD on fluoride ion ¹⁹F NMR relaxation can be monitored using one-dimensional experiments if an internal reference is present in the assay solution. To illustrate, the effects of CuZnSOD on ¹⁹F NMR spectra of solutions containing F⁻ and tfa are presented in Figure 1. Panel A in this figure shows the spectrum in the absence of enzyme, panel B shows the spectrum of the same solution but with ~10⁻⁷ M CuZnSOD added. Both spectra were acquired in one transient using a CPMG pulse sequence [32] with a 100 ms relaxation delay. These spectra demonstrate that SOD causes diminution of the peak intensity of the fluoride resonance relative to that of the tfa resonance. Inhibiting the NMR relaxation activity of the enzyme restores the intensity of the fluoride resonance.

A variety of polyphenolic organic compounds were tested for their abilities to inhibit ¹⁹F NMR relaxation by CuZnSOD. In an initial screen of seven natural products (apigenin, biochanin a, coumestrol, coumestrol dimethyl ether, quercetin, genistein, and daidzein), only quercetin: 1) affected a diminution of the ¹⁹F NMR relaxation rate. We note that the loss of activity did not occur in the absence of 1, nor does it appear to result from non- specific interaction governed by hydrophobicity; Overnight incubation of enzyme with more hydrophobic compounds such as apigenin; 2) had no effect on the relaxation activity of the enzyme. Subsequently, we measured the effects of a series of related compounds, including a variety of flavonoid natural products and lower molecular weight species. The effects of the compounds on the NMR relaxation rate were strongly dependent on pH, molecular structure, compound concentration, and dissolved oxygen. Structures of these compounds and further details of their interactions with the enzyme in aerobic solution are presented in the Supplemental section of this manuscript.

The effects of pH on the NMR activity were consistent with activity requiring deprotonation of a phenol group, 1 was less potent at pH 7 than at pH 8, and inhibitory activity dropped off even more significantly under acidic conditions. The pH dependence should not be surprising since flavonols typically have pK_a values around 7, 9, and 11 [33] - [37] and precipitate from neutral or acidic solutions [15] . To further probe the pH dependence, we measured the effects of small molecules including benzoic acid and ester derivatives on NMR relaxation by the

enzyme. Results of these experiments indicated that: 1) deprotonation of a carboxylic acid was not sufficient to induce inhibitory activity; 2) phenolic deprotonation is required; and 3) deprotonation of the enzyme at high pH renders it more susceptible to reaction. These results are described in greater detail in Supplemental Section 5.7.

The effects of twelve flavonoids and nine additional low molecular weight compounds on CuZnSOD NMR activity in aerobic aqueous solution were measured at pH 8.0. Inhibition of the NMR activity under these conditions depended strongly on compound structure; flavonols having hydroxyl groups at the 3, 3’ and 4’ positions were more effective than lower molecular weight polyphenols or flavonoids lacking any of these groups. Representative results are shown in Figure 2 for inhibition of the NMR activity by the flavonol, myricetin (3).

As shown in Figure 3, enzyme activity (calculated as described in the experimental section) was decidedly non-linear with polyphenol concentration. For many of the polyphenols, the behavior was consistent with aggregation limiting the availability of the active species. In most cases, the inhibitory behavior was well modeled by assuming a monomer/dimer equilibrium, with the monomer being the active species; The trend line in Figure 3 represents behavior expected for such a case. The concentration dependence of the fluorescence spectra of several flavonols supported this hypothesis. From the concentration dependence of the NMR activity inhibition data and the fluorescence data, it was possible to develop a structure/activity relationship for NMR activity inhibition by polyphenols. Inhibition of NMR activity by flavonols required hydroxyl groups at the 3, 3’ and 4’ positions. More detailed descriptions of our fluorescence results and of how NMR activities varied with compound structure are provided in the Supplemental Section.

Since the 3’ and 4’ hydroxyl groups constitute a catechol group on the flavonoid B ring, and since catechols (including 1 [38] ) form stable copper complexes [39] we considered the possibility that the catechol might chelate

the active site metal ion. Examples of species that inhibit CuZnSOD by interacting with the active site copper ion include low molecular weight anions (like cyanide and azide [40] - [44] ) and chelating agents like dithiocarbamates [45] - [48] . We note that catechol was among compounds reported to inhibit CuZnSOD by interacting with the active site copper [49] . The association equilibrium constant reported for copper binding by 1 (log(K) = 9.45 [50] ), however, is below the threshold reported for inhibition of SOD by metal binding (log(K) = 12.6) [49] .

While the low molecular weight CuZnSOD inhibitor diethyldithiocarbamate (DDC) acts by sequestering Cu²⁺ from the enzyme active site [48] , our results indicate that methyl-dihydroxybenzoate (MDHB, 4) does not act by this mechanism. In these experiments, we determined the concentrations of low molecular weight copper in enzyme samples after incubation in the presence and absence of 4. Copper concentrations were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). Low molecular weight fractions were isolated using Millipore YM-10 centrifugal concentration devices. Since the YM-10 device retains species larger than 10,000 Daltons, copper detected in the elutant should only arise from low molecular weight species. In these experiments, concentrations of copper in the low molecular weight fractions were not significantly affected by pre-incubated with the NMR inhibitor 4. In the 4 treated sample, 21% of the initial copper eluted as low molar mass material while 18% of copper in the control sample eluted. We conclude that the loss of NMR activity could not be attributed to the compound sequestering the metal from the enzyme.

To evaluate the feasibility of an inhibition mechanism where the compounds bound the metal in the active site, we used the Molecular Operating Environment (MOE, Chemical Computing Group, Montreal, Canada) suite of programs to dock the flavonoids to the 1PU0 structure of CuZnSOD. While these experiments indicated that the active site could accommodate the flavonols, most poses generated by docking calculations did not present the flavonol in contact with the enzyme copper atom. While it was possible to generate the proposed active site by tethering the flavonol B ring to the copper via the 3’ and 4’ hydroxyl groups, energy minimization did not return significantly stabilized structures. Thus, the docking results did not help rationalize the observed structure activity relationship.

To investigate the effects of the compounds on the active site copper, we measured the EPR spectrum of the oxidized enzyme in the presence and absence of two low MW polyphenols (4, MDHB and 5, MTHB) at pH 8 and 11. Representative EPR spectra collected at pH 8 are presented in Figure 4. In the absence of polyphenol, EPR spectra of CuZnSOD are consistent with the reported spectra at both pH 8 and 11, (g = 2.28, A = 178 [51] ). As shown in Figure 4(a), addition of 10 mM 4 (MDHB) to the enzyme results in a spectrum with sharp features that resemble those in the spectrum of Cu²⁺-MDHB (prepared from CuCl₂ and presented for comparison in the upper trace). Comparing the signal intensities in the spectra of CuZnSOD obtained before addition of 4 to that obtained 24 hours later, it appears that the total Cu²⁺ concentration is not grossly affected by the compound.

While the similarity of the spectrum to that of the Cu²⁺ complex of 4 suggests coordination, the centrifugal concentration experiments described above indicate that copper is not lost from SOD under these conditions. Thus, 4 does appear to coordinate copper in the active site of the protein.

In contrast to the effects of 4, addition of 10 mM 5 (MTHB) at pH 8 (Figure 4(b)) has less of an effect on the features of the spectrum but caused the resonance intensity to diminish with time. After 24 hour incubation with 10 mM 5, the Cu²⁺ resonance was nearly unobservable. We have verified that 5 reduces free Cu²⁺ at both pH 8 and 11 with loss of EPR signal. The increased ability of 5 (relative to 4) to reduce Cu²⁺ to Cu⁺ has been observed previously by EPR spectroscopy [52] . The difference in activities most likely reflects the difference in the redox potentials of the two compounds (E_pa values for 4 and 5 are 0.380 and 0.293 V, respectively) [39] . At pH 11, addition of either 4 or 5 to CuZnSOD resulted in a diminution of the Cu²⁺ signal intensity with time and a yellowing of the solution, indicating oxidation of the polyphenol (data not presented) [53] - [56] . In the case of 5, a sharp feature consistent with the resonance of a semiquinone radical (g = 2.003 [56] ) was transiently observed in the EPR spectrum (data presented in Supplemental section, part 5.10.). Sharp features were also observed at frequencies consistent with resonances of the Cu²⁺ complex of 5 at this pH.

The effects of oxygen on inhibition of the NMR activity were studied using either 1 mM 5 (MTHB) or 50 µM 1 (quercetin) (concentrations where ~50% inhibition of the NMR activity is observed in aerobic solution). In contrast to the behavior in aerobic environment, overnight incubation of CuZnSOD with either 1 or 5 under inert atmosphere resulted in complete loss of NMR activity. The activity versus time profiles for these experiments is presented in Figure 5. Sparging the completely inhibited samples with O₂ resulted in a nearly total restoration of NMR relaxation activity. These results are inconsistent with the possibilities that inhibition of the enzyme NMR activity involves reaction with oxygen radicals generated by aerobic oxidation of the organic compounds or with the products of such an oxidation.

Our results suggest that redox reactions (Equation (2) and Equation (3)) account for NMR activity inhibition.

Copper(II) salts will oxidize catechols [57] and are known to catalyze their aerobic oxidation to quinones [58] . The redox hypothesis predicts that the degree of apparent inhibition induced by a given reducing agent in aerobic solution would reflect the steady state concentrations of oxidized and reduced enzyme. Since the reactions in Equation (2) and Equation (3) appear to proceed to completion, and since Equation (3) is independent of the identity of the polyphenol, the SAR for the aerobic inhibition of NMR activity should reflect differences in the forward rate constants for Equation (2) for the different flavonols. These rates, in turn, are expected to reflect the differences in redox potentials of the compounds. A log/log plot of equilibrium constants calculated from published redox potentials versus IC₅₀ values from our NMR experiments (Figure 6) shows that the two parameters correlate. While a rigorous analysis would require substantially more data, the results presented in Figure 6 qualitatively support the hypothesis.

Since the Cu⁺ form of the enzyme is an intermediate in the enzymatic reaction, the redox hypothesis predicts that reaction with the polyphenols would not affect the ability of the enzyme to catalyze superoxide dismutation. To test the redox competence of the inhibited enzyme, we measured the effect of 1 on the steady state concentration of oxidized CuZnSOD under redox cycling conditions. Rigo et al. [25] [26] , demonstrated that treating the oxidized enzyme with superoxide causes the NMR relaxation activity of CuZnSOD to decrease by about half, as a steady state mixture of the reduced and oxidized enzyme is established. The position of the steady state was reported to vary for different batches of the enzyme. We used a mixture of phenozine methosulfate (pms) and NADH to generate superoxide [59] and monitored its effect on fluoride relaxation by CuZnSOD. While 1 is known to react with superoxide, the rate constant for this reaction is much lower than that for reaction of superoxide with CuZnSOD (5 × 10⁴ versus 2 × 10⁹ M⁻¹∙s⁻¹, respectively) [60] [61] . Thus, the interference of 1 with the reactions of superoxide with the enzyme should be minimal. Results of this experiment are shown in Figure 7. The open triangles show the effects of superoxide on NMR relaxation by the enzyme in the absence of 1. The filled diamonds show the effects of 10 µM 1 on the reaction. The filled diamonds show that, prior to the addition of pms/NADH, the NMR relaxation activity of the 1 containing sample was ~67% that of the control. After addition of pms/NADH, the sample containing 1 lost relaxation activity, although at a lower rate than the control sample. Eventually the control and 1 containing samples reached the same relaxation rates. This result suggests that CuZnSOD forms the same steady state mixture of reduced and oxidized enzyme whether 1 is present in the solution or not. Thus, inhibition of the enzyme NMR relaxation activity by flavonoids appears to arise from reduction of the enzyme to give a product that is inactive in the NMR assay but retains its ability to catalyze superoxide dismutation.