Modification of pig heart lipoamide dehydrogenase by cupric ions.

The insertion of a second disulfide bridge into native pig heart lipoamide dehydrogenase, requires two Cu-2+ ions for each catalytic center inactivated under anaerobic conditions. During inactivation, both metal atoms become reducible by their juxtaposition to the two participating cysteine residues and may be removed as the Cu+-chelates of neocuproine and bathocuproinesulfonate, leaving an additional disulfide bridge on the protein. Inactivation does not require the presence of oxygen, but when substoichiometric levels of copper are used under aerobic conditions the slow regeneration of Cu-2+ becomes rate-limiting. The course of aerobic inactivation is markedly biphasic at 0 degrees using 2 Cu-2+/FAD, with 30% of the total change completed rapidly, followed by a much slower phase. Both the extent of the fast phase and the rate of the second phase are enhanced by increasing levels of Cu-2+, but are relatively unaffected when the Cu-2+/FAD ratio is maintained at 2 and the protein concentration is varied. The enzyme affords several binding sites for Cu-2+ at pH 7.8, and it is suggested that competition between these sites during the initial statistical distribution of metal ions may explain this biphasic behavior.     

Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart.

A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.     

Nitroreductase activity of heart lipoamide dehydrogenase.

A novel reaction catalysed by lipoamide dehydrogenase is described. In the presence of NADH, lipoamide dehydrogenase reduces the nitro group of 4-nitropyridine and 4-nitropyridine N-oxide. The elution profiles from a DEAE-cellulose column for the dehydrogenase and nitroreductase activities are identical. Chemical modifications of critical amino acid residues suggest that the two activities share a common catalytic domain. Nitro reduction catalysed by lipoamide dehydrogenase was monitored spectrophotometrically and chromatographically. The major product from the enzymic reduction of 4-nitropyridine was isolated and characterized structurally as NN-bis(pyridinyl)hydroxylamine, which is formed presumably via 4-hydroxyaminopyridine in a four-electron redox reaction.     

Multifunctionality of lipoamide dehydrogenase: Role of histidine residues in reductase reaction

Rose bengal sensitizes photoinactivation of lipoamide dehydrogenase from pig heart to a constant residual reductase activity resulting from specific destruction of histidine residues. The rate of sensitized photoinactivation is pH dependent and is associated with an ionizable group with pK 6.6 ± 0.2. All steady-state kinetic parameters are markedly reduced by photooxidation. Spectroscopic studies indicate the contribution of oxidized flavin/dithiol to the half-reduced form of the photooxidized enzyme. The proton magnetic resonance spectrum of lipoamide dehydrogenase shows resolved histidine C₂ proton peak at δ9.18 ppm and a shoulder at δ9.23 ppm. The shoulder protons are eliminated by the sensitized photooxidation and shifted upfield on deprotonation. At high pH, the characteristic Faraday A term also disappears. These observations suggest that the essential histidine stabilizes the nascent thiolate via the ion pair formation to facilitate the reductase reaction catalyzed by lipoamide dehydrogenase.     

Comparative studies of glutathione reductase and lipoamide dehydrogenase

1. Glutathione reductase and lipoamide dehydrogenase are structurally and mechanistically related flavoenzymes catalyzing various one and two electron transfer reactions between NAD(P)H and substrates with different structures. 2. The two enzymes differ in their coenzyme and functional specificities. Lipoamide dehydrogenase shows higher coenzyme preference while glutathione reductase displays greater functional specificity. 3. Binding preference of the two flavoenzymes for nicotinamide coenzymes is demonstrated by 31P-NMR spectroscopy. 4. The presence of arginines in glutathione reductase which is inactivated by phenyl glyoxal, is likely to be responsible for the NADPH-activity of glutathione reductase. 5. The substrate binding sites of the two enzymes are similar, though their functional details differ. 6. The active-site histidine of glutathione reductase functions primarily as the proton donor during catalysis. While the active-site histidine of lipoamide dehydrogenase stabilizes the thiolate anion intermediate and relays a proton in the catalytic process.     

Titration studies on the active sites of pig heart lipoamide dehydrogenase and yeast glutathione reductase as monitored by the charge transfer absorbance

Macroscopic pKa values associated with the influence of pH on the visible spectrum of 2-electron reduced pig heart lipoamide dehydrogenase and yeast glutathione reductase have been determined by monitoring changes in the principal flavin band near 460 nm and the charge transfer band at 540 nm. The ionization of at least three active site amino acid side chains can influence the spectra over the range of pH studied: the two nascent thiols (interchange thiol and electron transfer thiol) and the histidine residue which acts as the base catalyst in lipoamide dehydrogenase and the acid catalyst in glutathione reductase thiol-disulfide interchange reactions. These systems are analogous to, but more complex than, those in glyceraldehyde-3-phosphate dehydrogenase and papain where a single thiol and a histidine residue in a relatively apolar milieu form a thiolate-imidazolium ion pair which is favored over the thiol-imidazole prototropic tautomer. In an effort to more nearly mimic the papain titrations, the macroscopic pKa values were determined on reduced glutathione reductase which had been monoalkylated with iodoacetamide under conditions known to favor the reaction of the interchange thiol by at least 10 to 1 (Arscott, L. D., Thorpe, C., and Williams, C. H., Jr. (1981) Biochemistry 20, 1513-1520). Like papain and glyceraldehyde-3-phosphate dehydrogenase, alkylated glutathione reductase showed two macroscopic pKa values, at pH 3.7 and pH 9.1, and by analogy, these were associated primarily with the thiol and the imidazole, respectively. Results with the native enzymes depended on the wavelength monitored. Glutathione reductase had pKa values at 4.8, 7.1, and 9.2 when monitored at 540 nm and 5.1 and 8.2 when monitored at 462 nm. Lipoamide dehydrogenase had pKa values at 4.4 and 8.7 when monitored at 529 nm and 3.9, 7.0, and 9.3 when monitored at 455 nm.     

An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.     

Measurement of the oxidation-reduction potentials for two-electron and four-electron reduction of lipoamide dehydrogenase from pig heart.

The oxidation-reduction potential, E2, for the couple oxidized lipoamide dehydrogenase/2-electron reduced lipoamide dehydrogenase has been determined by measurement of equilibria of these enzyme species with lipoamide and dihydrolipoamide or with oxidized and reduced azine dyes. E2 is -0.280 V at pH 7, and deltaE2/deltapH is -0.06 V in the pH range 5.5 to 7.6. Values for E1, the oxidation-reduction potential for the couple 2-electron reduced enzyme/4-electron reduced enzyme, were obtained from measurements of the extent of dismutation of 2-electron reduced enzyme to form mixtures containing oxidized and 4-electron reduced enzyme. E1 is -0.346 V at pH 7, and deltaE1/deltapH is -0.06 V in the pH range 5.7 to 7.6. Spectra of oxidized enzyme and 4-electron reduced enzyme do not show variations with pH over this range, but the spectrum of the 2-electron reduced enzyme is pH-dependent, with the molar extinction at 530 nm changing from 3250 M-1 cm-1 at pH 8 to 2050 M-1 cm-1 at pH 5.2. The pH-dependent changes which are observed in the absorption properties of the 2-electron reduced enzyme are consistent with the disappearance of a charge transfer complex between an amino acid side chain and the oxidized flavin at the lower pH values, with the apparent pK of the side chain at pH 5. It has been suggested that the 530 nm absorbance of 2-electron reduced enzyme is due to a charge transfer complex between thiolate anion and oxidized flavin, and we propose that the thiolate anion is stabilized by interaction with a protonated base. The thermodynamic data predict that the amount of 4-electron reduced enzyme formed when the enzyme is reduced by excess NADH will be pH-dependent, with the greatest amounts seen at low pH values. These data support earlier evidence (Matthews, R.G., Wilkinson, K.D., Ballou, D,P., and Williams, C.H., Jr. (1976) in Flavins and Flavoproteins (Singer, T.P., ed) pp. 464-472; Elsevier Scientific Publishing Co., Amsterdam) that the role of NAD+ in the NADH-lipoamide reductase reaction catalyzed by lipoamide dehydrogenase is to prevent accumulation of inactive 4-electron reduced enzyme by simple reversal of the reduction of 2-electron reduced enzyme by NADH.     

Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase.

A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.     

Rapid relaxation processes in pig heart lipoamide dehydrogenase revealed by subnanosecond resolved fluorometry

The decay kinetics of the FAD-fluorescence in lipoamide dehydrogenase from pig heart have been reinvestigated using phase fluorometric methods and sophisticated laser pulse techniques. Both pulse and modulation methods lead to distinct heterogeneity in lifetimes. The two different techniques lead to good correspondence in the longer lifetime component of a biexponential decay model, whereas the more rapidly decaying component is distinctly shorter and has a larger amplitude using the phase technique with two available modulation frequencies (15 and 60 MHz). Lifetime measurements as a function of temperature and in the presence of D₂O instead of H₂O illustrate that the quenching of the FAD fluorescence in lipoamide. dehydrogenase is mainly dynamic in nature and that solvent comes into contact with the fluorophor. Mobility of the flavin itself, free and bound to the enzyme, has been measured by both differential polarized phase fluorometry and experimental fluorescence anisotropy decay after ps laser pulse excitation. By employing flavin models it has been shown that both techniques have ps time resolution. Measurements with the latter more direct method indicate a rapid subnanosecond motion of the FAD bound within the enzyme, only visible at temperatures lower than about 15°C, where the protein rotational diffusion is slowed down. The significance of rapid transient conformational fluctuations for catalysis is discussed with reference to recently developed insights reported in the literature.