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W Maret

Publications and source records attributed to W Maret.

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PubMed1987

Electronic absorption and EPR spectroscopy of copper alcohol dehydrogenase: pink, violet and green forms of a type 1 copper center analog.

Blue and non-blue states of the copper center in copper-substituted alcohol dehydrogenase (EC 1.1.1.1) can be attained by coenzyme binding and/or ligand binding to the copper ion. Copper alcohol dehydrogenase has been studied by electronic absorption, CD and EPR spectroscopy in the presence and absence of coenzyme. On the basis of previous work on blue (Type 1) copper proteins with a CuSS*N2 chromophore the assignment of charge transfer transitions in copper alcohol dehydrogenase is discussed. The latter contains a CuS2N(OH2) unit in the ligand-free protein and a CuS2N2 unit in the ternary complex with NAD+ and pyrazole. It is proposed that the energy of the charge transfer transitions can be used as a structural marker in combination with EPR data. A comparison is made between the spectroscopic properties of the ternary complex of copper alcohol dehydrogenase and the copper centers in stellacyanin and cytochrome-c oxidase (CuA) in order to test the validity of recent structural models of the type CuS2N2, i.e., a cupric ion coordinated to two thiolate ligands. Finally, a close resemblance between the electronic absorption spectra of copper alcohol dehydrogenase and those of other variants of Type 1 copper centers such as the 'unusual' copper center of nitrous oxide reductase is noted as an indication of similar coordination environments.

Alcohol Dehydrogenase↗
PubMed1987

The influence of anions and inhibitors on the catalytic metal ion in Co(II)-substituted horse liver alcohol dehydrogenase.

1H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition 13C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the beta-CH2 protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The 1H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3- and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD+ only the resonances of the beta-CH2 protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1986

Influence of anions and pH on the conformational change of horse liver alcohol dehydrogenase induced by binding of oxidized nicotinamide adenine dinucleotide: binding of chloride to the catalytic metal ion.

The conformational change of horse liver alcohol dehydrogenase induced by binding of NAD+ was studied by electronic absorption spectroscopy using cobalt as a spectroscopic probe in the active site. The complex of the enzyme with NAD+ exists in an acidic and an alkaline form. The transition between the two forms proceeds through several intermediates and is controlled by an apparent pKa of 6.9. Only at pH values below this pKa can a complex between enzyme, NAD+, and Cl- be formed. The spectral changes indicate that chloride displaces the cobalt-bound water molecule in a tetracoordinate structure. We conclude that a negative charge at the active site is necessary to stabilize the closed conformation of the enzyme in the presence of NAD+. Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1986

Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites.

The zinc-deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13-ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1-anilinonaphthalene 8-sulphonic acid is bound with comparable affinity. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc-deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 2,2'-Bipyridine, which chelates the active-site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc-depleted enzyme. The stopped-flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc-depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His-67, bound to the zinc ion, or the zinc-liganding water molecule in the case of the native enzyme (pK close to 9), or the free His-67 residue in the case of the zinc-deficient enzyme (pK about 8).

2,2'-Dipyridyl↗
PubMed1985

The interaction of catalytic metal ions and ionizing groups in equilibrium studies and in transient intermediates of metal-substituted alcohol dehydrogenases.

The step of ternary complex interconversion in the reaction catalyzed by horse liver alcohol dehydrogenase has been resolved into five distinct molecular species with the aid of metal-substitution studies in combination with rapid-scanning spectrophotometry. A correlation with electronic absorption spectra at equilibrium provides structural insights into these intermediates. In contrast to NADH, NAD+ only leads to a conformational change of the protein when a negative charge has been created in the vicinity of the catalytic metal ion. This paper presents also a reevaluation of previous assignments of catalytically important groups in the light of some recent results.

Alcohol Oxidoreductases↗
PubMed1983

Resonance Raman spectra of copper(II)-substituted liver alcohol dehydrogenase: a type 1 copper analogue.

Liver alcohol dehydrogenase (LADH) with copper in place of the catalytic zinc has recently been proposed to contain a type 1 site analogous to that in "blue" copper proteins. Resonance Raman spectra for the copper-substituted enzyme, Cu(II) X LADH, and its binary complexes with reduced nicotinamide adenine dinucleotide (NADH) and pyrazole support this viewpoint. These spectra have two dominant features: a sharp peak at approximately 415 cm-1, which is believed to be associated with vibration of the single histidine ligand, and a broader, asymmetric band at approximately 350 cm-1, whose components are assigned predominantly to vibrational modes of the two cysteinate ligands. The high frequency of these transitions, which is reminiscent of the blue copper proteins, is ascribed to the tetrahedral nature of the metal site that produces unusually short Cu-S bonds and coupled vibrational modes. Solvent exchange with H218O reveals no contribution to the resonance Raman spectrum of the water molecule, which is a metal ligand in free Cu(II) X LADH; however, the spectrum of the binary complex with pyrazole has several new peaks attributable, in part, to pyrazole ligation. The strong similarity among the vibrational spectra demonstrates that the Cu(II) environment in alcohol dehydrogenase maintains its near-tetrahedral geometry in the various enzyme derivatives. The resonance Raman spectrum of Ni(II) X LADH is close to that of Cu(II) X LADH and suggests a similar tetrahedral site. The Raman spectra presented here together with available optical and EPR data indicate that Cu(II) X LADH belongs to the type 1 copper classification and, thus, can provide new insights into this unusual coordination geometry.

Alcohol Dehydrogenase↗
PubMed1983

An EPR study of the blue copper center in horse liver alcohol dehydrogenase.

Active-site specifically reconstituted Cu2+ horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) shows optical and EPR spectra similar to those of native blue copper (Type 1) proteins. EPR spectra at different temperatures and frequencies reveal a heterogeneity of the copper center: a minor 'non-blue' species with axial line shape (g parallel = 2.16, g perpendicular = 2.04; A parallel = 100 x 10(-4) cm-1), which accounts for approximately 10% of the total copper and is not accessible to ligands and a major blue species with rhombic line shape (g1 = 2.21, g2 = 2.06, g3 = 2.03, A1 = 50 x 10(-4) cm-1, A2 = 30 x 10(-4) cm-1, A3 = 76 x 10(-4) cm-1), which is accessible to ligands and participates in redox reactions. The major blue species in cupric horse liver alcohol dehydrogenase is metastable, since it is reduced in a process markedly accelerated in the presence of oxygen or hydrogen peroxide. In addition, the reduction depends on the presence of exogenous metal ligands or coenzymes. Whereas the binary complex enzyme-NAD+ is even more susceptible to bleaching than the free enzyme, the cupric center is stable towards bleaching in the binary complex enzyme-NADH. In the discussion the redox properties and coordination chemistry of Cu2+ in horse liver alcohol dehydrogenase and copper proteins are compared.

Alcohol Dehydrogenase↗
PubMed1983

Neutral metal-bound water is the base catalyst in liver alcohol dehydrogenase.

The catalytic role of the active site metal-water complex in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) is investigated on the basis of a comparative analysis of the pH dependence of steady-state kinetic parameters of the native and active-site-specific Co2+-reconstituted enzyme and on the basis of assignment of the coordination environment of the Co2+ by electron paramagnetic resonance methods. The pH dependence of the kinetic parameters for the oxidation of benzyl alcohol reveals two ionizations (pK1 approximately equal to 6.7; pK2 approximately equal to 10.6) that govern kcat and belong to the ternary enzyme-NAD+-alcohol complex and two ionizations (pK1' approximately equal to 7.5; pK2' approximately equal to 8.9) that govern kcat/Km and belong to the binary enzyme-NAD+ complex. The ionizations pK2 and pK2' decrease by 0.5-1 pK alpha unit upon replacement of the active site Zn2+ by Co2+. A similar metal ion dependence of pK2 and pK2' is observed for the oxidation of 2-propanol. We attribute these ionizations to a metal-bound water molecule. The zero-field splitting energy of the Co2+ in the binary enzyme-NADH complex and the ternary enzyme-NADH-CF3CH2OH complex is approximately equal to 22 cm-1, indicative of a pentacoordinate species. Binding of a water molecule to the metal ion as the fifth ligand in the ternary enzyme-NADH-CF3CH2OH complex is confirmed on the basis of magnetic interactions of H2(17)O with Co2+. The results indicate that the active site metal ion in catalytically competent ternary enzyme-coenzyme-substrate complexes is pentacoordinate and is ligated by a neutral water molecule in the physiological pH range. We suggest that the neutral metal-bound water molecule serves as the base catalyst for proton abstraction in alcohol oxidation.

Alcohol Dehydrogenase↗
PubMed1981

X-ray absorption edge spectroscopy of Co(II)-binding sites of copper- and zinc-containing proteins.

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and [...-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase greater than stellacyanin greater than superoxide dismutase greater than or equal to carbonic anhydrase.

Alcohol Oxidoreductases↗
PubMed1981

Metal ion substitution at the catalytic site of horse-liver alcohol dehydrogenase: results from solvent magnetic relaxation studies. 2. Binding of manganese(II) and competition with zinc(II) and cadmium(II) ions.

The interaction of Mn2+ aquo ions with native horse-liver alcohol dehydrogenase demetalized specifically at the catalytic sites has been investigated by studying the magnetic field dependence and time dependence of the magnetic spin-lattice relaxation rate of solvent water protons. We find no detectable binding of Mn2+ ions to the catalytic sites in times on the order of hours; however, we do find that these ions bind to the enzyme at two previously unreported types of sites: one, characterized by a low dissociation constant (0.01 mM at pH 7.7, 5 degrees C), low relaxivity, and a stoichiometry of one per two catalytic sites, and a second, with a high dissociation constant (1.5 mM at pH 7.7, 5 degrees C) and high relaxivity. The stoichiometry of the second type of site could not be determined because of the relatively weak bindng of Mn2+ ions to these sites. Both Zn2+ and Cd2+ ions bind to the newly found tight-binding sites, displacing Mn2+ ions and thereby altering the relaxation rates of solvent protons. By monitoring the return to equilibrium of these altered rates, we find that Zn2+ ions enter the catalytic sites from the new tight-binding sites with an on-rate of approximately 0.1 M-1 s-1. It is not clear whether binding to these new sites is an obligatory intermediate for reintroduction of Zn2+ ions into the catalytic sites, but a small excess of Zn2+ ions beyond one per monomer causes the protein to precipitate. Cd2+ ions, by contrast, enter the catalytic sites at least 1 order of magnitude more rapidly than do Zn2+ ions, a rate too rapid to observe by our techniques. However, once the catalytic sites are filled, Cd2+ ions displace Mn2+ ions at the new sites as do Zn2+ ions.

Alcohol Dehydrogenase↗
PubMed1981

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: optical spectra of binary and ternary complexes with coenzymes, coenzyme analogues, substrates, and inhibitors.

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M-1 cm-1), 407 nm (3500 M-1 cm-1), 505 nm (300 M-1 cm-1), 570 nm (approximately equal to 130 M-1 cm-1), and 680 nm (approximately equal to 80 M-1 cm-1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry. The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH greater than 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal on which acts as a Lewis acid in both substrate coordination and catalysis.

Alcohol Dehydrogenase↗
PubMed1980

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions.

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.

Alcohol Oxidoreductases↗
PubMed1979

Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernlöhr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.

Alcohol Oxidoreductases↗
PubMed1978

Agarose-bound horse-liver alcohol dehydrogenase. Dependence of molecular properties and activity on coupling conditions.

1. Spectroscopic methods for protein and active-site determination with the same sample of immobilised horse liver alcohol dehydrogenase have been developed. 2. The influence of pH, active-site protection of the soluble enzyme and protein concentration on coupling of alcohol dehydrogenase with cyanogen-bromide-activated Sepharose has been investigated. In phosphate buffer (pH 8.0) products with over 90% active-site retention have been synthesized. The binary complex alcohol-dehydrogenase . NADH gives a preparation with the same active-site content but a lower apparent specific activity compared to the unprotected enzyme. Increase in protein concentration yields products with the same active-site content relative to bound protein but the apparent specific activity is decreased. 3. The great similarity in spectroscopic properties of soluble and immobilised enzyme, as well as of their ternary complexes, shows that no significant conformational change has taken place during immobilisation. 4. Exchange of the non-catalytic Zn2+ against Co2+ yields a hybrid Sepharose--Co2Zn2-alcohol-dehydrogenase with over 90% active-site retention during metal exchange. The absorption spectra of the soluble and immobilised hybrid are identical.

Alcohol Oxidoreductases↗