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• W2080223471 abstract "Submicromolar zinc inhibits α-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the α-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441–13447). Lipoamide dehydrogenase, a component of the α-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD+. This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD+. The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn2+(K i ∼0.15 μm) in both directions. Steady-state kinetic studies revealed that Zn2+ competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn2+ with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn2+ accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 ± 0.02 μm. Activation is a consequence of Zn2+binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn2+ on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn2+ can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism. Submicromolar zinc inhibits α-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the α-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441–13447). Lipoamide dehydrogenase, a component of the α-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD+. This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD+. The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn2+(K i ∼0.15 μm) in both directions. Steady-state kinetic studies revealed that Zn2+ competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn2+ with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn2+ accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 ± 0.02 μm. Activation is a consequence of Zn2+binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn2+ on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn2+ can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism. dihydrolipoyl dehydrogenase (lipoamide dydrogenase) dithiothreitol oxidized lipoamide (dl-6,8-thioctic acid amide) superoxide dismutase tris[hydroxymethyl]aminomethane reactive oxygen species A number of reports suggest that mobilization of intracellular Zn2+ may play a role in cellular toxicity following ischemia-reperfusion injury (1Choi D.W. Koh J.Y. Ann. Rev. Neurosci. 1998; 21: 347-375Crossref PubMed Scopus (681) Google Scholar, 2Choi D.W. Yokoyama M. Koh J. Neuroscience. 1988; 24: 67-79Crossref PubMed Scopus (204) Google Scholar, 3Koh J.-Y. Suh S.W. Gwag B.J. He Y.Y. Hsu C.Y. Choi D.W. Science. 1996; 272: 1013-1016Crossref PubMed Scopus (943) Google Scholar, 4Frederickson C.J. Hernadez M.D. McGinty J.F. Brain Res. 1989; 480: 317-321Crossref PubMed Scopus (307) Google Scholar). Aberrant Zn2+regulation has also been noted in Alzheimer's disease brain (5Deibel M.A. Ehmann W.D. Markesbery W.R. J. Neurol. Sci. 1996; 143: 137-142Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 6Danscher G. Jensen K.B. Frederickson C.J. Kemp K. Andreasen A. Juhl S. Stoltenberg M. Ravid R. J. Neurosci. Meth. 1997; 76: 53-59Crossref PubMed Scopus (162) Google Scholar, 7Cornett C.R. Markesbery W.R. Ehmann W.D. Neurotoxicology. 1998; 19: 339-345PubMed Google Scholar). Elevated intracellular Zn2+ has been associated with loss of mitochondrial membrane potential, production of reactive oxygen species, and cell death (3Koh J.-Y. Suh S.W. Gwag B.J. He Y.Y. Hsu C.Y. Choi D.W. Science. 1996; 272: 1013-1016Crossref PubMed Scopus (943) Google Scholar, 8Manev H. Kharlamov E. Uz T. Mason R.P. Cagnoli C.M. Exp. Neurol. 1997; 146: 171-178Crossref PubMed Scopus (125) Google Scholar, 9Sensi S.L. Yin H.Z. Carriedo S.G. Rao S.S. Weiss J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2414-2419Crossref PubMed Scopus (362) Google Scholar). Recently our laboratory reported that Zn2+ is a potent inhibitor of α-ketoglutarate-stimulated mitochondrial respiration (10Brown A.M. Kristal B.S. Effron M.S. Shestopalov A.I. Ullucci P.A. Sheu K.-F.R. Blass J.P. Cooper A.J.L. J. Biol. Chem. 2000; 275: 13441-13447Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The effect of Zn2+ on respiration was attributed to inhibition of the α-ketoglutarate dehydrogenase complex (10Brown A.M. Kristal B.S. Effron M.S. Shestopalov A.I. Ullucci P.A. Sheu K.-F.R. Blass J.P. Cooper A.J.L. J. Biol. Chem. 2000; 275: 13441-13447Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Preliminary analysis of individual subunit activities of α-ketoglutarate dehydrogenase complex indicated that the lipoamide dehydrogenase (LADH)1 displayed the greatest susceptibility to Zn2+ inhibition. LADH belongs to the family of flavin-disulfide oxido-reductases (11Williams Jr., C.H. Muller F. Chemistry and Biology of Flavoenzymes. III. CRC Press, Boca Raton, FL1992: 121-212Google Scholar), which include glutathione reductase and thioredoxin reductase. LADH is an essential component of the multienzyme NADH-generating complexes α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, branched-chain ketoacid dehydrogenase, and also glycine decarboxylase in plants (12Bourguignon J. Merand V. Rawthorne S. Forest E. Douce R. Biochem. J. 1996; 313: 229-234Crossref PubMed Scopus (38) Google Scholar). In these multienzyme complexes, LADH catalyzes the transfer of reducing equivalents from the bound lipoate of the neighboring transferase subunit to NAD+. LADH also catalyzes the reduction of free lipoic acid by NADH and thus helps to maintain the reducing environment of mitochondria required for defense against reactive oxygen species and formation of electron-rich structures such as iron-sulfur clusters. Dihydrolipoic acid, the reduced form of lipoic acid, is a potent antioxidant that scavenges reactive oxygen species such as superoxide, peroxyl radicals, hypochlorous acid, and nitric oxide. Dihydrolipoic acid may also regenerate other antioxidants such as vitamins C and E and glutathione through redox cycling (13Packer L. Trischler H.J. Wessel K. Free Radic. Biol. Med. 1997; 22: 359-378Crossref PubMed Scopus (635) Google Scholar). Preliminary human studies indicate the efficacy of lipoic acid in treatment of cerebral ischemia-reperfusion, excitotoxic amino acid brain injury, mitochondrial dysfunction, and other disorders involving free radical processes (13Packer L. Trischler H.J. Wessel K. Free Radic. Biol. Med. 1997; 22: 359-378Crossref PubMed Scopus (635) Google Scholar, 14Tirosh O. Sen C.K. Roy S. Kobayashi M.S. Packer L. Free Radic. Biol. Med. 1999; 26: 1418-1426Crossref PubMed Scopus (91) Google Scholar). LADH (EC 1.8.1.4) is a homodimeric molecule, each subunit (∼52 kDa) contains two catalytic centers. FAD is responsible for NAD+/NADH reduction/oxidation and the redox-active disulfide interacts with the dihydrolipoyl cofactor that is covalently linked to the acetyl transferase component of each dehydrogenase complex. The reaction with lipoic acid/lipoamide is reversible. When the reaction occurs in the same direction as within the multienzyme complex, it is referred to as the forward reaction. When it proceeds in the direction of lipoic acid reduction by NADH, it is referred to as the reverse reaction. NAD++L(SH) 2→NADH+LS2forward NADH+LS2→NAD++L(SH) 2reversewhere LS2 and L(SH)2 are lipoic acid and dihydrolipoic acid, respectively. LADH also exhibits catalytic activity in the reduction of one- and two-electron organic and inorganic acceptors with NADH and in the reduction of molecular oxygen as shown below. NADH+DCPIPOX→NAD++DCPIPreddiaphorase NADH+2[Fe(CN) 6] 3−→NAD++2[Fe(CN) 6] 2−electron­transferase NADH+O2→NAD++H2O2oxidaseThe lipoamide dehydrogenase reaction follows a ping-pong mechanism with the intermediate formation of the two-electron-reduced enzyme (15Massey V. Gibson Q.H. Veeger C. Biochem. J. 1960; 77: 341-351Crossref PubMed Scopus (119) Google Scholar) (Scheme FS1). The latter can exist in forms containing both reducing equivalents on FAD (EFADH2), both reducing equivalents on thiols (EFAD(SH)2), or in the form, referred to as the charge-transfer complex, with one electron on a reduced thiol and the other electron shared between FAD and thiolate (EFADHSSH). The charge-transfer complex is the major form of two-electron-reduced enzyme and has a characteristic absorption shoulder at 530 nm distinguishing it from other enzyme forms (16Sahlman L. Williams Jr., C.H. J. Biol. Chem. 1989; 264: 8033-8038Abstract Full Text PDF PubMed Google Scholar). Early studies concluded that under anaerobic conditions of equimolar reduction with NADH the form with two reduced thiols contributes 22% and the charge-transfer complex contributes 34% to the total two-electron-reduced pig heart enzyme. Thus, the major form (44%) contains fully reduced FAD (17Veeger C. Massey V. Biochim. Biophys. Acta. 1962; 64: 83-100Crossref PubMed Scopus (29) Google Scholar). More recently it has been proposed that NAD+ release directly yields the charge-transfer complex. The enzyme form with fully reduced FAD is not thought to be significantly populated (18Maeda-Yorita K. Russell G.C. Guest J.R. Massey V. Williams Jr., C.H. Biochemistry. 1991; 30: 11788-11795Crossref PubMed Scopus (20) Google Scholar). Based upon the current understanding of the enzymatic mechanism (11Williams Jr., C.H. Muller F. Chemistry and Biology of Flavoenzymes. III. CRC Press, Boca Raton, FL1992: 121-212Google Scholar) (Scheme FS1) and structure of LADH (19Toyoda T. Kobayashi R. Sekiguchi T. Koike K. Koike M. Takenaka A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 982-985Crossref PubMed Scopus (8) Google Scholar, 20Toyoda T. Suzuki K. Sekiguchi T. Reed L.J. Takenaka A. J. Biochem. 1998; 123: 668-674Crossref PubMed Scopus (26) Google Scholar, 21Mattevi A. Schierbeek A.J. Hol W.G.J. J. Mol. Biol. 1991; 220: 975-994Crossref PubMed Scopus (151) Google Scholar), our working hypothesis was that the target for Zn2+ inhibition is the reduced catalytic disulfide (see Fig. 1). To test this hypothesis, we have undertaken detailed studies on the effect of Zn2+ on LADH-catalyzed reactions. The present paper demonstrates that submicromolar levels of free Zn2+ strongly inhibit lipoamide dehydrogenase activity by interaction with the catalytic disulfide. Zn2+ binding also stimulates production of reactive oxygen species by LADH. Steady-state and anaerobic transient kinetics experiments are presented that establish the mechanism of these phenomena.Figure 1A schematic view of LADH active center demonstrating a hydrophobic channel leading lipoic acid to the redox-active disulfide. The redox disulfide active site is formed at the interface between monomers A and B. Key catalytic amino acid residues are labeled in bold. Residues of monomer B are shown in italic. Double bonds correspond to carbonyl bond of the backbone. The crystal structure of dimeric LADH from pyruvate dehydrogenase complex ofBacillus stearothermophilus (accession number EBD1) was used. The drawing was prepared using MSI WebLab ViewerPro.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NADH, NAD+, α-lipoamide (dl-6,8-thioctic acid amide),dl-dithiothreitol (DTT), tris(hydroxymethyl)aminomethane (Tris), H2O2, ZnCl2, and EDTA were from Sigma. All reagents were “SigmaUltra grade,” if available. Dimethyl sulfoxide (Me2SO) was from Fisher Scientific (Fair Lawn, NJ), HCl (ACS grade) was from J. T. Baker, Inc. (Phillipsburg, NJ). All solutions were prepared using distilled, deionized water with >15 MΩ/cm resistance. LADH from porcine heart from Sigma was additionally purified by fast protein liquid chromatography gel-filtration on a Superdex 200 column (Amersham Biosciences, Inc.) equilibrated with 0.2 mTris-HCl buffer, pH 7.5. LADH concentration was determined spectrophotometrically (ε455 = 11,300m−1 cm−1 (22Matthews R.G. Williams Jr., C.H. J. Biol. Chem. 1976; 251: 3956-3964Abstract Full Text PDF PubMed Google Scholar)). Catalase from bovine liver (EC 1.11.1.6; 18,600 units/mg) and superoxide dismutase from bovine erythrocytes (EC 1.15.1.1; 3,500 units/mg) were obtained from Sigma and used without further purification. All spectrophotometric measurements were performed in a 96-well plate reader (SpectraMax Plus, Molecular Dynamics, Sunnyvale, CA) with a 200-μl reaction mixture per well at 20 °C. The absorbance of NADH (ε340 = 6,220m−1 cm−1 (23Popov V.O. Gazarian I.G. Egorov A.M. Berezin I.V. Biochim. Biophys. Acta. 1985; 827: 466-471Crossref Scopus (13) Google Scholar)) was monitored. The light path was 0.43 cm. All reactions were run in 50 mmTris-HCl, pH 7.5. Each set of experiments was performed in triplicate. All data reported are as the means ± S.E. Reverse reaction: the final concentrations of reagents were 30 nm LADH, 0.05–0.2 mm LA, 0.01–0.1 mm NADH, 0.1–10 μm ZnCl2. To avoid severe substrate inhibition by NADH (17Veeger C. Massey V. Biochim. Biophys. Acta. 1962; 64: 83-100Crossref PubMed Scopus (29) Google Scholar) the concentration range for NADH was chosen to be lower than that for LA. Forward reaction: the final concentrations were 30 nm LADH, 0.02–0.1 mm reduced LA, 0.2–2 mmNAD+, 1–12 μm ZnCl2 (final Me2SO concentration in individual wells was <1%). The study of this reaction in the presence of zinc is complicated because Zn2+ can be coordinated by thiols, such as reduced LA and DTT. In the presence of excess DTT (1 mm) no inhibition by Zn2+ was observed. To avoid chelating Zn2+ with excess of thiol reagents, LA in Me2SO was reduced with equimolar DTT dissolved in water and then diluted into a buffer solution saturated with argon in order to avoid oxidation. Aliquots of NAD+ and zinc solutions were placed into the wells, and the reaction was initiated by the addition of argon-saturated buffer containing reduced lipoamide and enzyme. Oxygen consumption was measured in a “Strathkelvin Instruments” (Glasgow, UK) multichannel apparatus model SI-928 with a Clark type electrode. Reactions were monitored in thermostatted (28 °C) glass chambers fitted with special glass stoppers that contained a small injection hole (Gilson) and equipped with a magnetic stirrer (“Instech” 2060, Plymouth Meeting, PA). The electrode was calibrated in the range 0–0.240 mm oxygen immediately prior to the experiments. Oxygen concentration was varied in the range 20–240 μmby bubbling buffer with argon to displace oxygen. The final concentrations of reagents were in the range 0.1–0.2 μmenzyme, 0.01–0.5 mm NADH, 0.01–0.25 NAD+, and 1–40 μm ZnCl2. Data analyses were performed using “Strathkelvin Instruments” software SI version 2.1. Each experiment with the oxygen electrode was performed in triplicate. The rate constants were calculated as an average from three independent sets of experiments. Anaerobic stopped-flow measurements were performed in duplicate using a High-Tech SF-61 stopped-flow rapid scan spectrophotometer (High Tech Scientific, Salisbury, Wiltshire, UK) installed in an anaerobic glove box operating under N2 with less than 1 ppm O2. The temperature was controlled at 22 °C with a Techne-400 circulator (Techne (Cambridge) Ltd., Duxford, Cambridgeshire, UK) with an external cooler. The enzyme solution and substrate powder were placed into the sealed serum vials and deoxygenated 1 h before being placed into the glove box. The 0.05m Tris-HCl buffer, pH 7.5, used in all experiments was deoxygenated overnight in the glove box. NADH (0.75 mm) and ZnCl2 (1.875 mm) stock solutions were prepared anaerobically under N2 in the same buffer. Initial experiments were performed in rapid-scan mode with a xenon lamp. The enzyme stock solution (15 μm) was shot against rising concentrations of NADH (7.5–60 μm) to determine the NADH concentration exhibiting the highest absorbance at 530 nm, which is characteristic of the two-electron-reduced LADH in the form of a charge-transfer complex (16Sahlman L. Williams Jr., C.H. J. Biol. Chem. 1989; 264: 8033-8038Abstract Full Text PDF PubMed Google Scholar). Based on this determination, a 1.5 molar excess of NADH over the enzyme was used to generate the maximum content of a charge transfer complex, which then was shot against varying concentrations of ZnCl2 (7.5–75 μm). The absorbance time-curves at 530 nm were fitted to exponential functions using a least-squares minimization program supplied by High-Tech Scientific. Since Tris is known to bind Zn2+ with a binding constant of ∼10−4m (24Dawson R.M.C. Elliot D.C. Elliot W.H. Jones K.M. Data for Biological Research. Oxford Science Publications, Oxford University Press, Oxford, UK1986: 410Google Scholar), the 50 mm Tris buffer used in our experiments is expected to sequester most of Zn2+ added. The thermodynamic binding constant reported for Tris binding of Zn2+ must be corrected for pH and ionic strength before free Zn2+ can be calculated. The concentration of free Zn2+ available for enzyme binding was determined experimentally. In calculation of Zn2+ inhibition constants, a denominator of 20 was used to recalculate the available zinc concentration from the total added one (see data for details). No changes in the mechanism of LADH reaction were found within the range of 0.05–1 μm free Zn2+concentrations. The reaction continues to obey a ping-pong mechanism. In the reverse reaction, Zn2+ and LA compete for one and the same enzyme form. The addition of rising concentrations of zinc results in increased slopes and an unchanged intercept in double-reciprocal plots of 1/v versus 1/[LA] (not shown). Competitive inhibition is confirmed by a clear single intersection point in the Dixon plot (see Fig. 2 A), corresponding to the inhibition constant of ∼0.15 μm. With respect to NADH, Zn2+ is an uncompetitive inhibitor showing parallel lines both in double-reciprocal plots and in the Dixon plot (not shown). Thus, zinc is unable to react with oxidized enzyme. The data fit the equationEo /v = 1/k o + 1/k NADH[NADH] + 1/k LA[LA](1 + [Zn]/K Zn) with k NADH = (2 ± 0.5) × 106m−1s−1, k LA = (3.0 ± 0.1) × 105m−1 s−1,k o′ = 300 ± 10 s−1, andK Zn = 0.15 ± 0.05 μm. Lipoamide and Zn2+ compete for the same enzyme form. It is possible that Zn2+ either attacks the charge-transfer complex, which then rearranges into a form with both thiols participating in Zn2+ binding or directly binds to the form with reduced thiols, shifting the equilibrium toward the above enzyme form. We can speculate that the catalytic His residue paired with a highly conserved Glu residue also participate in Zn2+coordination (Fig. 1). The inhibition mechanism can be described by Scheme FS1 with Zn2+ binding the two-electron-reduced enzyme in the form of a charge-transfer complex or in the form with reduced thiols. In the forward reaction of NAD+ reduction by reduced lipoamide, Zn2+ demonstrates uncompetitive inhibition with respect to lipoamide (not shown). This type of inhibition suggests that Zn2+ does not react with oxidized enzyme, which is consistent with the mechanism proposed in Scheme FS1. However, double-reciprocal plots of 1/v versus1/[NAD+] indicate that Zn2+ affects both slope and intercept (Fig.2 B). If the reaction follows the mechanism presented in Scheme FS1, the dependence of the forward reaction rate in double-reciprocal plots should display pure competitive inhibition with respect to NAD+ in accordance with Equation 1. Eo/v=1/k−1+1/k−3+(k−3+k4)/k−3k−4[LAred]Equation 1 +(1+[Zn]/Ki)](k−1+k2)/k−1k−2[NAD]Even if one accounts for the possible presence of oxidized lipoamide, the effect of Zn2+ in accordance with Scheme FS1will be seen only on the apparent rate constant for NAD+(slope), but not on intercepts as shown in Equation 2. Eo/v=1/k−1+1/k−3+(k−3+k4)/k−3k−4[LAred]+(1+k3[LA]/k−3Equation 2 +k3k4[LA]/k−3k−4[LAred]+[Zn]/Ki)(k−1+k2)/k−1k−2[NAD]Thus, the model presented in Scheme FS1 is not consistent with the experimental data. The effect of Zn2+ on both the rate constant for NAD+ and the rate constant for a unimolecular step suggests that Zn2+ binds to an enzyme form other than the one directly interacting with NAD+. Taking into account the existence of different forms of the two-electron-reduced enzyme (18Maeda-Yorita K. Russell G.C. Guest J.R. Massey V. Williams Jr., C.H. Biochemistry. 1991; 30: 11788-11795Crossref PubMed Scopus (20) Google Scholar), we can introduce a unimolecular interconversion step into SchemeFS1 to get Scheme FS2, which satisfactorily describes the inhibition by Zn2+ for both reverse and forward reactions (see also TableI). The reverse and forward reactions exhibit equal values for the inhibition constant KZn = K i(1 +k −3/k 3), within experimental error (see Table I). The trend toward a larger inhibition constant determined for the forward reaction from Dixon plot (Fig.2 C, 0.22 ± 0.05 μm) compared with that determined for the reverse reaction (Fig. 2 A, 0.15 ± 0.05 μm) may originate from Zn2+ chelation by reduced lipoamide.Table ISteady-state kinetic parameters of Zn2+ inhibition of LADH reactionReverse reactionExperimental equation:E o/v = 1/k o′ + 1/k NADH[NADH] + 1/k LA[LA](1 + [Zn]/K Zn)Equation derived for Scheme FS2:E o/v = 1/k 2+ 1/k 5 + 1/k 3 + (1 +k −3/k 3 + [Zn]/K 1)(k −4 +k 5)/k 4 k 5[LA] + (k −1 +k 2)/k 1 k 2[NADH]First order rate constant,k o′ =k 2 k 3 k 5/(k 2+ k 3 + k 5),s −1300 ± 10Rate constant for NADHk NADH =k 1 k 2/(k −1 +k 2), m−1 s−1(2.0 ± 0.5) × 106Rate constant for LAk LA =k 4 k 5/(1 +k 3/k −3)(k −4+ k 5), m−1s−1(3.0 ± 0.1) × 105Inhibition constantK Zn = K i(1 +k −3/k 3), μm0.15 ± 0.05Forward reactionExperimental equation:E o/v = 1/k o′(1 + [Zn]/K Zn′), + 1/k NAD[NAD](1 + [Zn]/K Zn) + 1/k LA[LAred]Equation derived for Scheme FS2:E o/v = 1/k −1 + 1/k −4 + 1/k −3(1 + [Zn]/K i) + (k −4 +k 5)/k −4 k −5[LAred] + (1 + k 3/k −3 +k 3[Zn]/k −3 K i)(k −1+k 2)/k −1 k −2[NAD]First order rate constantk o′ =k −1 k −3 k −4/(k −1+ k −3 + k −4), s−130 ± 10Rate constant for reduced LAk LAred =k −4 k −5/(k −4+ k 5), m−1s−1(8.0 ± 1.0) × 104Rate constant for NAD+k NAD =k −1 k −2/(1 +k 3/k −3)(k −1+ k 2), m−1s−1(1.2 ± 0.2) × 104Inhibition constantK Zn(slope) = (1 +k −3/k 3)K i, μm0.22 ± 0.05K Zn′(intercept) = (1 +k −3/k −1 +k −3/k −4) K i, μm1.1 ± 0.2 Open table in a new tab The charge transfer complex is clearly distinguishable from the oxidized enzyme and two-electron-reduced enzyme with reducing equivalents on both thiols (11Williams Jr., C.H. Muller F. Chemistry and Biology of Flavoenzymes. III. CRC Press, Boca Raton, FL1992: 121-212Google Scholar) due to a characteristic shoulder at 530 nm. The effect of Zn2+ on the spectrum of the two-electron-reduced enzyme was studied by the stopped-flow technique under anaerobic conditions (Fig. 3). ZnCl2 addition to the charge transfer complex leads to rapid disappearance of the 530-nm shoulder and an increase in absorption at 445 nm (see inset A in Fig. 3). Thus, the Zn2+·LADH complex is spectrally equivalent to the oxidized enzyme form. The rate of the spectral change at 530 nm displays linear dependence on Zn2+ concentration (inset B in Fig. 3). This indicates that the experimentally measurable step is Zn2+binding not interconversion between the charge-transfer complex and the dithiol form (Scheme FS3). The determined rate constant for Zn2+ interaction with the two-electron-reduced enzyme is (3.7 ± 0.7) × 105m−1 s−1. This value is comparable with the rate constant for the two-electron-reduced form interaction with lipoamide determined from the steady-state kinetics (3.0 ± 0.1 × 105m−1 s−1). Thus, Zn2+successfully competes with lipoamide for this enzyme form. The non-zero intercept at the ordinate axis clearly points to the reversibility of the reaction and corresponds to the rate constant for the Zn2+-complex dissociation (Scheme FS3). The dissociation rate constant k −i is equal to 0.040 ± 0.005 s−1. The equilibrium constant for Zn2+ binding calculated on the basis of stopped-flow measurementsK i = k −i/k i = 0.11 ± 0.04 μm, which is close to the inhibition constants determined from steady-state kinetics (Table I). The ratio between the inhibition constants determined from steady-state data and the binding constant determined from transient kinetics allows to estimate the ratiok −3/k 3 as 1.0 ± 0.5. Thus, the contribution of the charge-transfer complex and the form with reduced two thiols to the total pool of two-electron-reduced enzyme are comparable within experimental error. Zn2+ binding is reversible. This has been demonstrated both by dilution of assay mixtures and by EDTA addition, which completely reversed all inhibitory effects (data not shown). In addition, gel-filtration of the Zn2+-inhibited assay mixture restored the initial catalytic activity (results not shown). Activation of molecular oxygen by flavoproteins, yielding hydrogen peroxide (dehydrogenases/transhydrogenases, oxidases) and superoxide radical (electron transferases such as flavodoxin), is a well known process (25Massey V. J. Biol. Chem. 1994; 269: 22459-22462Abstract Full Text PDF PubMed Google Scholar). After the flavoenzymes of the respiratory chain, the most abundant flavoprotein in the mitochondrial matrix (∼0.5% of matrix protein as calculated from Ref. 26Matuda S. Saheki T. J. Biochem. 1982; 91: 553-561Crossref PubMed Scopus (20) Google Scholar) is lipoamide dehydrogenase. Production of hydrogen peroxide and superoxide radical catalyzed by lipoamide dehydrogenase was first reported in 1955 (27Huennekens F.M. Basford R.E. Gabrio B.W. J. Biol. Chem. 1955; 213: 951-967Abstract Full Text PDF PubMed Google Scholar) and then confirmed in 1969 (28Massey V. Strickland S. Mayhew S.G. Howell L.G. Engel P.C. Matthews R.G. Schumanm M. Sullivan P.A. Biochem. Biophys. Res. Commun. 1969; 36: 891-897Crossref PubMed Scopus (299) Google Scholar). More recently, identification of reaction products revealed the ratio between superoxide and hydrogen peroxide to be 1:9 (29Bando Y. Aki K. J. Biochem. 1991; 109: 450-454Crossref PubMed Scopus (37) Google Scholar). However, no quantitative studies to determine the oxidase reaction mechanism have been reported. In the present work the formation of hydrogen peroxide as a major product was demonstrated by addition of catalase (Fig.4 A), which resulted in recovery of ∼50% of the consumed oxygen via disproportionation to O2 and H2O. Prior addition of catalase reduced the rate of oxygen consumption by half, providing further support that hydrogen peroxide is the major product (Fig. 4 B). Addition of SOD at the end of the reaction resulted in detectable O2recovery (Fig. 4, A and B) indicating the generation of superoxide anion radical, which is consistent with the published report (29Bando Y. Aki K. J. Biochem. 1991; 109: 450-454Crossref PubMed Scopus (37) Google Scholar). Zn2+ addition increased the rate of oxygen consumption, but did not change the character or distribution of the products formed (Fig. 4 A). The stimulatory effect of Zn2+ was observed in the presence of NAD+, although the overall reaction showed strong product inhibition (Fig. 4 A). At lower oxygen concentration product inhibition by NAD+ was even more pronounced, but the stimulatory effect of Zn2+remained clearly evident (Fig. 4 C). The dependence of the reaction rate on oxygen concentration in double-reciprocal plots shows parallel lines both in the absence and in the presence of Zn2+ (Fig.5 A). It is also evident that Zn2+ affects the rate constants toward NADH and oxygen in a different manner. There is little or no increase in the rate constant toward oxygen in the presence of Zn2+" @default.
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