Substrate specificity of copper-containing plant amine oxidases.

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.

Is the catalytic mechanism of bacteria, plant, and mammal copper-TPQ amine oxidases identical?

This short review is mostly concerned with the work carried out in Rome on the copper amine oxidase from bovine serum (BSAO). The first target was the copper oxidation state and its relationship with the organic cofactor. It was found that copper is not reduced on reaction with amines under anaerobic conditions or along the catalytic cycle and that it is not within bonding distance of the quinone cofactor. The copper stability in the oxidised state was supported by BSAO ability to oxidise benzylhydrazine, a slow substrate, in the presence of N,N-diethyldithiocarbamate (DDC) and by the substantial catalytic activity of Co(2+)-substituted BSAO. Parallel work established that only one subunit of the dimeric enzyme readily binds reagents of the carbonyl group. Flexible hydrazides with a long aromatic tail were found to be highly specific inhibitors, suggesting the presence of an extended hydrophobic region at the catalytic site. A study by stopped-flow transient spectroscopy and steady state kinetics led to the formulation of a simplified, yet complete and consistent, catalytic mechanism for BSAO that was compared with that available for lentil seedling amine oxidase (LSAO). As in other copper amine oxidases, BSAO is inactivated by H(2)O(2) produced in the catalytic reaction, while the cofactor is stabilised in its reduced state. A conserved tyrosine hydrogen-bonded to the cofactor might be oxidised.

Reaction of N,N-diethyldithiocarbamate and other bidentate ligands with Zn, Co and Cu bovine carbonic anhydrases. Inhibition of the enzyme activity and evidence for stable ternary enzyme-metal-ligand complexes.

The reactions with N,N-diethyldithiocarbamate (DDC) of zinc, cobalt and copper carbonic anhydrase from bovine erythrocytes were investigated. The native zinc enzyme was inhibited by DDC, but no removal of zinc could be detected even at a very high [ligand]/[protein] ratio. At identical pH values a larger inhibitory effect was found for the cobalt enzyme. The metal was removed by DDC from the protein at pH less than 7.0. No cobalt removal occurred at pH 10, where a stable ternary complex with the enzyme-bound Co(II) was detected. Its optical and EPR spectra are indicative of five-coordinate Co(II). The reaction of the Cu(II) enzyme with stoichiometric chelating agent was marked by the appearance of an electronic transition at 390 nm (epsilon = 4300 M-1 X cm-1). Metal removal from the copper enzyme readily occurred as the ligand was in excess over the metal, with parallel appearance of a band at 440 nm, which was attributed to the free Cu(II)-DDC complex. Also, in the case of the copper enzyme an alkaline pH was found to stabilize the ternary adduct with the diagnostic 390 nm band. EPR spectra showed that the ternary adduct is a mixture of two species, both characterized by the presence in the EPR spectrum of a superhyperfine structure from two protein nitrogens and by a low g parallel value, indicative of coordination to sulfur ligands. It is suggested that the two species contain the metal as penta- and hexacoordinated, respectively. Measurements of the longitudinal relaxation time, T1, of the water protons suggested that water coordination is retained in the latter case. Hexacoordination with retention of water is also proposed for the Cu(II) derivatives with the bidentate oxalate and bicarbonate anions, unlike the corresponding Co(II) derivatives, which are pentacoordinated. Different coordination of Co(II) and Cu(II) adducts may be relevant to the difference of activity of the two substituted enzymes.

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.

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.

Reconstitution of stellacyanin as a case of direct Cu(I) transfer between yeast copper thionein and 'blue' copper apoprotein.

It was of interest to examine whether yeast Cu-thionein could be used to transfer the thiolate bound copper directly into the copper binding site of 'blue' apoproteins which contain free thiol groups. In particular apo-stellacyanin was used in the present study and it was found to be able to accept Cu(I) from yeast Cu-thionein, without any detectable unspecific Cu(II) intermediate, both aerobically and anaerobically.

An electron spin resonance study of high spin forms of cobalt(II) bovine carbonic anhydrase.

The ESR spectra of bovine Co(II) carbonic anhydrase at 7 K at low and high pH and of the iodide derivative have been analyzed. The spectrum of the low pH form shows axial symmetry whilst that at high pH is rhombically distorted. This anisotropy is still more accentuated in the iodide derivative. The high pH (hydroxyl) form and the iodide derivative are thought to have a tetracoordinate trigonal pyramidal structure, with a fifth more distant axial ligand. The low pH form is consistent with a pseudotetrahedral geometry previously postulated.

A resonance Raman study of native stellacyanin and its Ni(II) derivative. On the origin of the 450-nm electronic absorption.

The resonance Raman spectra of native stellacyanin and its Ni(II) derivative has been investigated. Raman intensity as a function of the exciting frequency shows minima at about 440--460 nm. Moreover, the resonance Raman spectra of the Ni(II) derivative indicate similar symmetry and nature of ligands, namely, one cysteine and at least one histidine.Optical electronegativity of the ligand involved in the intense visible absorption band of native stellacyanin and the corresponding transition of the Ni(lI) derivative confirm this assumption. The origin of the 450 nm band is also discussed.

Effects of laser radiation on Rhus vernicifera laccase, Type 2 Cu-depleted laccase, and stellacyanin.

Laser irradiation in the 450 nm region brings about irreversible changes in the copper sites of Rhus vernicifera lactase and its type 2 Cu-depleted derivative. The absorption band at 614 nm disappears after _ 2 hr of irradiation with a 200 mW laser beam; the amount of the paramagnetic detectable copper does not decrease, indicating no reduction of these types of copper. No apparent rearrangement of the protein backbone occurs, as ultaviolet dichroic spectra of the enzyme before and after the irradiation do not show appreciable differences. Stellacyanin is insensitive to laser radiation at any wavelength.

Electron transfer kinetics between Rhus vernicifera stellacyanin and cytochrome c (horse heart cytochrome c and Pseudomonas cytochrome c551).

The electron transfer reactions between Rhus vernicifera stellacyanin and either horse heart cytochrome c or Pseudomonas aeruginosa cytochrome c551 were investigated by rapid reaction techniques. The time course of electron transfer is monophasic under all conditions, and thus consistent with a simple formulation of the reaction. Both stopped-flow and temperature-jump experiments yield equilibrium constants in reasonable agreement with values calculated from the redox potentials. The differences in reaction rate between the two cytochromes and stellacyanin are discussed in terms of the Marcus theory.

Characterization of the haemoglobin-mediated inhibition of the enzymatic activity of bovine serum amine oxidase.

Haemoglobin has been previously identified as responsible for the decreased enzymatic activity of copper bovine serum amine oxidase (BSAO) in suspensions of human or bovine hemolyzed erythrocytes [Marcocci, L., Pietrangeli, P., Befani, O., Mavelli, I., & Mondovi', B. (1991b) Life Chem. Report, 9, 171-177]. This is confirmed by present results on bovine methaemoglobin. Bovine globin and horse skeletal muscle mioglobin showed a similar inhibiting ability, but neither bovine serum albumin nor cytochrome c inhibited BSAO activity under the same experimental conditions. The inhibitory effect of bovine haemoglobin was dependent on pH only at high buffer ionic strength. It was highest in physiological conditions (PBS) where haemoglobin acted as a reversible non competitive inhibitor of BSAO activity, with apparent Ki of 0.5 mM at 37 degrees C. The inhibition was unaffected by partial BSAO deglycosylation (40% of glucidic residues removed) but decreased when haemoglobin lysine groups were derivatised using citraconic anhydride. A possible molecular mechanism underlying the inhibitory effect is discussed.