BIOLOGICAL TRACE ELEMENT RESEARCH 2, 159-174 (1980)

Copper (II) Complex-Catalyzed Oxidation of NADH by Hydrogen Peroxide PHILLIP C. CHAN*

AND LEO KESNER

Department of Biochemistry, State University of New York Downstate Medical Center, Brooklyn, New York 11203 Received January 7, 1980; Accepted February 29, 1980

Abstract Among various metal ions of physiological interest, Cu 2§ is uniquely capable of catalyzing the oxidation of NADH by H202. This oxidation is stimulated about fivefold in the presence of imidazole. A similar activating effect is found for some imidazole derivatives (l-methyl imidazole, 2-methyl imidazole, and N-acetyl-L-histidine). Some other imidazole-containing compounds (L-histidine, L-histidine methyl ester, and L-carnosine), however, inhibit the Cu2§ peroxidation of NADH. Other chelating agents such as EDTA and L-alanine are also inhibitory. Stoichiometry for NADH oxidation per mole of H202 utilized is 1, which excludes the possibility of a two-step oxidation mechanism with a nucleotide free-radical intermediate. About 92% of the NADH oxidation product can be identified as enzymatically active NAD § D20, 2,5-dimethylfuran, and 1,4-diazabicyclo [2.2.2]-octane have no significant effect on the oxidation, thus excluding tO2 as a mediator. Similarly, OH. is also not a likely intermediate, since the system is not affected by various scavengers of this radical. The results suggest that a copper-hydrogen peroxide intermediate, when complexed with suitable ligands, can generate still another oxygen species much more reactive than its parent compound, H202. Index Entries: Copper (II) complex, as catalyst in H202 oxidation of NADH; catalyst, Cu (II) in H202 oxidation of NADH; hydrogen peroxide, Cu (II) catalysis of NADH oxidation by; NADH, oxidation by Cu (II) complex and H202; oxidation, of NADH by Cu (II) catalyzed H202.

9 1980 The Humana Press Inc. All rights of any nature whatsoever reserved. 0163 4 9 8 4 / 80 / 0600-0159503.20

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CHAN AND KESNER

Introduction By comparison with some of the other oxygen species of physiological interest, hydrogen peroxide is not very reactive as an oxidant. However, in several biological systems hydrogen peroxide has been implicated as a precursor of various cytotoxic agents; tO2 (1), OH. (1-5), and some other yet unidentified species (6, 7). The results in this study, using NADH as a substrate model, indicate that the oxidative activity of hydrogen peroxide in neutral pH range can be enhanced considerably by suitable ligand-copper complexes. Inhibitor studies have excluded either 102 or OH. as a mediator in this system, thereby indicating that certain Cu2+-H202 complexes may play a significant role among the other well known reactive oxygen species in biological systems. Under drastic experimental2+ conditions" " in either acidic media or at high temperatures, Cu has been shown to catalyze hydrogen peroxide oxidation of various compounds: saturated and unsaturated fatty acids (8, 9), benzoic acid (10), catechol (11), collagen (12), and amino acids (12). Peroxidative activities in copper-containing proteins, ceruloplasmin (13,14), hemocyanin (15), and superoxide dismutase (16) have also been demonstrated. In a series of studies on the copper complex-catalyzed decomposition of H202, Sharma and Schubert (17-20) found that various ligands, including imidazole, can potentiate the catalatic activity of Cu 2§ Superoxide dismutase activities have also been found in several amino acid copper complexes (21, 22). In this study we tested some of these complexes for their peroxidative activity towards NADH. We found that only imidazole and some of its derivatives are capable of stimulating Cu2§ oxidation of NADH to NAD § by H202 at neutral pH. Many of the other complexes with catalatic and/or superoxide dismutase activities were either inhibitors or without effect on NADH oxidation. Burton and Lamborg (23) found that in a slightly alkaline medium, NADH, NAD § and nicotinamide riboside were oxidized by high concentrations of Cu 2§ and H202.

Materials and Methods NADH was obtained from P-L Biochemicals, 30% H202 and CuSO4 from Baker Chemical Co., catalase and lactate dehydrogenase from Boehringer Mannheim Corp., and 99.8% D20 and imidazole from Sigma Chemical Co. Imidazole was recrystallized from benzene. Highly purified samples of CuSO4 (99.99%) from Apache Chemicals, Inc. and Research Organic/Inorganic Chemical Corp. were also tested. In this system no detectable difference was found between these samples and the "Baker Analyzed" reagent.

Cu (II)-CATALYZEDOXIDATIONOF NADH BY HK)2

161

Oxidation of N A D H was carried out in a Cary 14 Spectrophotometer. A typical reaction mixture contained 10 m M imidazole, 10 m M sodium phosphate, pH 7.2, 1 0 / z M CuSO4, 0.2 m M NADH, and 2.7 m M H202 added at zero time. The total volume was 3.0 mL and the temperature was 30~ C. All other components, when added, were also adjusted to pH 7.2. The rate of oxidation was calculated from the recorded changes in absorbance at 340 nm, using a molar extinction of 6.2 • 10 3 M -1 cm -~. Concentration of H202 was determined according to the procedure of Allen et al. (24).

Results

Copper-Catalyzed Oxidation of NADH by H202 Table 1 shows that in the presence of 10 mM imidazole and 10 m M phosphate, the oxidation of N A D H by H:O2 could be stimulated over 38fold by 10/.~M Cu 2+ ions. About half the activating activity was observed with cuprous ions, which may have resulted from partial conversion of Cu + to Cu 2§ in the reaction mixture with an excess of H202. All the other metal ions tested exhibited either little or no effect on N A D H oxidation.It is of interest to note that under these conditions no detectable Fenton reaction (25) as presented in Reaction 1 was observed with FeSO4. Fe 2+ + H202 --" Fe 3~ + OH- + OH"

(1)

Schellenberg and Hellerman (26) have demonstrated the oxidation of N A D H by OH" produced in Reaction 1 in a lower pH medium with a lower TABLE 1 Effect of Various Metal Ionsa Addition, 10/aM Control (no addition) CuSO4 Cu2Cl2 COC12 ZnSO4 HgCl2 AgNO3

FeSO4 Pb (NO3)3 CdCl2

NADH oxidation,/~M/min 0.5 19.2 9.8 1.5 1.1 0.8 0.8

0.7 0.6 0.6

=Other compounds tested and found to be without activating effect included: FeCI3, MnSO4, Cr(NO~)3, NiSO4, MgCI2, Na2SeO3, Na2SeO4, CaCI2, and (NH4)2MoO4.

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C H A N AND KESNER

HzO2 concentration. We have confirmed their observation using their conditions, and have also found that at pH 5 the Fenton reaction was not inhibited by addition of 10 m M imidazole. Even when 2 m M A D P was added to increase the solubility of Fe :§ no significant oxidation was observed. This may be explained by the excess of H202, which can also serve as an OH" scavenger (25): H202 + OH"

--

+ HO2"

H20

(2) The experiments in Fig. 1 illustrate the roles of Cu 2§ and HzO2 in N A D H oxidation. Experiment (a) is a typical run with the standard reaction mixture, It can be seen that a decrease in A~40 was observed as soon as H202 was added at zero time. There was a lag period with a somewhat slower 1.4

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TIME (MINUTES) FIG. I. Effect of EDTA and catalase. Reaction mixtures for the 4 experiments were identical at zero time, including 0.2 mM NADH, I0 #MCuSO4 (30 nmol), I0 m M sodium phosphate, I0 mM imidazole, and 2.7 mM H202 added at zero time, pH 7.2. The total volume was 3.0 mL. The absorbance baselines for Experiments b, c, and d were raised slightly to avoid overlapping. (a) Control run; (b) 500 nmol EDTA in 5 #L added at Arrow 1,500 nmol CuSO4 in 5/.tL added at Arrow 2, another 500 nmol CuSO4 added at A r r o w 3; (c) 500 nmol EDTA added at Arrow 4, 500 nmol CuS 04 added atA rrow 5, 60 nmol CuSO4 in 5/.t L added at A r r o w 6; (d) 20 /ag catalase in l0/.zL added at A r r o w 7.

Cu (II)-CATALYZED

OXIDATION

OF NADH

BY H202

163

reaction rate during the first 2 min, and this was followed by a zero-order reaction rate. This was the rate used for all comparison studies. As will be shown later, upon prolonged incubation almost all of the NADH can be oxidized. In Fig. 1, Experiment (b), when 500 nmol EDTA in 10/.tL was added ,the oxidation of NADH was halted instantly. Addition of an equal amount of CuSO4 did not initiate a change. When a large excess of CuSO4 was added, the remaining NADH was oxidized rapidly as shown by the sharp drop in A340.

Experiment (c) shows that the total concentration of CuSO4 can be adjusted to resume the initial rate of NADH oxidation. These results suggest that the inhibitory effect on EDTA owes mainly to its ability to chelate Cu 2§ rather than as a scavenger for reactive oxygen species. Experiment (d) shows that when H202 is decomposed by catalase, oxidation of NADH was quickly terminated. In another experiment, to test the effect of an equivalent amount of protein, 20/.tg of bovine serum albumin was added during the reaction. No detectable change in the rate of NADH oxidation was observed. Figure 2 shows that the rate of NADH oxidation is proportional to the concentration of Cu 2§ within the range tested. The effects of the concentrations of H202 and NADH are more complicated and neither demonstrated saturation kinetics within the range studied. Figure 3 represents the Arrhenius plot for the reaction. The energy of activation calculated from these results equals to 13.9 kcal. The temperature in all the other experiments in this study were carefully regulated at 30 + 0.5~ The overall reaction of NADH oxidation by H202 indicates an uptake of H +. This was verified by an increasing pH during the reaction when it was carried out in a medium of low buffer capacity. Figure 4, however, shows that the rate of oxidation increases rapidly with increasing pH, especially above pH 8. This suggests that a high OH- concentration is favorable for the rate-limiting step, similar to that observed by Sharma and Schubert in their study of H202 decomposition catalyzed by an imidazole-Cu2§complex (18). They proposed the ionization of H202 as the main influencing factor for the pH profile. Other factors such as the ionization of imidazole and phosphate may also contribute to the effect of pH.

Ligands of Copper Complexes Imidazole and its derivatives are known to be effective ligands for copper complex formation. Most of these complexes are capable of catalyzing 9 9 9 9 2+ decomposalon of H202 (17-19). When these hgands were tested m the Cu catalyzed oxidation of NADH by H202 (Table 2), only imidazole, 1-methyl imidazole, 2-methyl imidazole, and N-acetyl-L-histidine were found to have

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added. This was expected since these substances behave similarly to imidazole in stimulating the copper-catalyzed oxidation of NADH. In the molecule of L-carnosine, although the amino group of the histidyl residue is acylated, the amino group of the/3-alanyl residue can also serve as a ligand for Cu 2§ and thereby contribute to the inhibition of NADH oxidation. On the other hand, when an equimolar amount of methylamine was added to th.e imidazole complex, it did not bring about the same effect as histamine, which possesses the same quantities of imidazole and primary amino groups. These results indicate that either imidazole or an imidazole group is capable of forming active complexes with cupric ion. If an amino group is also present in the same molecule, however, the complex with mixed ligands is no longer reactive (e.g., L-histidine and L-carnosine). The presence of a carboxyl group in the same molecule (e.g., N-acetyl-L-histidine) has no effect on the peroxidative activity. This is further substantiated in Fig. 5, which shows that the activity profiles with varying concentrations of either imidazole or N-acetyl-L-histidine are similar. In order to eliminate the possibility that a ligand Cu 2+ complex may inhibit the oxidation of NADH by rapidly depleting H202 in the system,

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FIG. 4. Effect of pH on the rate of oxidation. Same amounts of buffer components (10 mMimidazole and 10 mM sodium phosphate) were present in each experiment. The p H of the reaction mixture was adjusted with either NaOH or HCI, and measured immediately before the reaction. TABLE 2 Comparison of Various Ligands with Imidazole a Addition, 5 m M Control Imidazole l-Methyl imidazole N-Acetyl-L-histidine 2-Methyl imidazole L-Carnosine Methylamine L-Histidine methyl ester L-Histidine L-Alanine EDTA

Rate of NADH oxidation, #M/min 4.4 21.0 20.6 18.4 12.9 4.0 2.7 0.0 0.0 0.0 0.0

~The reaction mixture contained 0.2 mM NADH, 10 #M CuSO4, l0 mM sodium phosphate and additions as indicated, nH 7.2.

167

Cu (II)-CATALYZED OXIDATION OF NADH BY H202

TABLE 3 Effects of Various Ligands in the Presence of Imidazole~ Percent inhibition by second ligand

Rate of NADH oxidation, #M/min

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20.8 0.0 0.5

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1.0

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Copper (II) complex-catalyzed oxidation of NADH by hydrogen peroxide.

Among various metal ions of physiological interest, Cu(2+) is uniquely capable of catalyzing the oxidation of NADH by H2O2. This oxidation is stimulat...
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