The role of copper binding in the conformation of stellacyanin

doi:10.1016/0003-9861(74)90263-X

Archives of Biochemistry and Biophysics

Volume 161, Issue 1, March 1974, Pages 291-296

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Abstract

The binding of Cu²⁺ to apostellacyanin occurs in two steps. The first step consists of a fast equilibrium reaction involving binding of copper to the protein in a non-native, though specific way, as shown by electron paramagnetic resonance measurements. All the spectroscopic properties of native stellacyanin are recovered in a slower monomolecular process (k = 7.5 × 10⁻³ sec⁻¹ at 25 °C) characterized by high activation energy (ΔH_a = 22 kcal mole⁻¹) and low activation entropy (ΔS_a = 3.0 cal deg⁻¹ mole⁻¹). The second step parallels a conformational change of the copper-bound protein molecule. A large difference of the tyrosyl residues pKs is found between holo- and apostellacyanin. In the latter the tyrosyl residues appear to be more exposed to solvent perturbations. Ammonia or monovalent anions such as N₃⁻, SCN⁻, and Cl⁻ have a catalytic effect on the second step of the reaction, roughly proportional to their first binding constant to aqueous copper. It is suggested that they may compete for a non-native bond of the copper to the protein, thus rendering the conformational change easier.

The effect of Ag³ and Hg²⁺ on the recombination reaction with copper is discussed in terms of conformation of the metal-bound protein.

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Studies on plantacyanin. IV. Reconstitution with Cu-thionein, oxidation by cytochrome oxidase and autooxidation in the presence of cardiolipin
1991, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Cu-thionein isolated from cucumber roots was used for reconstitution of plantacyanin from cucumber. The rate of the copper transfer from Cu-thionein to apoplantacyanin was found to depend on pH, ionic strength and concentrations of the proteins. The rate of reconstitution with Cu-thionein was 10-times higher than with copper ions. No intermediate was observed during reconstitution with Cu-thionein. The incubation of oxidized holoplantacyanin with Cu-thionein or apothionein brings about the reduction of plantacyanin copper. This process, however, was found to be slow as compared to the rate of copper transfer from Cu-thionein to apoplantacyanin. Cytochrome oxidase from heart mitochondria was detected to possess some plantacyanin oxidase activity with the turnover number 5 min⁻¹. The activity of the enzyme towards plantacyanin as well as with cytochrome c as a substrate was established to be lipid and ionic strength-dependent, and it was inhibited by CN⁻ and N₃⁻. Lineweaver-Burk plots show that the inhibitory effect of ionic strength on plantacyanin oxidase activity is connected with changes of Michaelis constant rather than of the maximal rate. Plantacyanin which is known to be very resistant towards many cationic, anionic and nonionic detergents, becomes, as well as cytochrome c, autooxidable in the presence of cardiolipin.
Zn2+ regulation of ornithine transcarbamoylase. I. Mechanism of action
1990, Journal of Molecular Biology
Ornithine transcarbamoylase catalyzes the formation of l-citrulline from carbamoyl phosphate and l-ornithine. The anabolic enzyme from Escherichia coli is composed of three identical subunits and resembles, in both primary and quaternary structures, the catalytic unit of aspartate transcarbamoylase. However, ornithine transcarbamoylase has no regulatory subunits. Although this enzyme does not bind its substrates co-operatively, fluorescence spectroscopic experiments show that zinc adds allosterically to the free enzyme. The metal binding process is dictated by deprotonation of an enzymic group with a pK_a < 7. The saturation binding curve of the metal ion is sigmoidal and yields a Hill coefficient of 1.6 at pH 8.5 and 25 °C. In the absence of substrates, zinc further promotes a slow enzyme isomerization, which occurs with a first-order rate constant of 7 min⁻¹; thus, the metal is a slow, tight-binding inhibitor. The isomerized enzyme is inactive and contains three zinc ions. When the enzyme is first bound with carbamoyl phosphate, steady-state kinetic assays reveal that zinc again binds co-operatively to the binary enzyme complex, with a Hill coefficient of 1.5, but the metal ion now behaves simply as a classical, reversible inhibitor; it is competitive against l-ornithine, non-competitive against carbamoyl phosphate, and induces no enzyme isomerization. However, as a result of the competition between zinc and l-ornithine for the same site on the enzyme, the l-ornithine saturation curve becomes sigmoidal. Displacement of the allosteric zinc from the enzyme by l-ornithine is the cause of co-operative addition of this substrate. The combined results suggest that zinc regulates ornithine transcarbamoylase via two routes: (1) as an allosteric cofactor of the substrate-bound enzyme in mediating site-site interactions; and (2) as a slow, tight-binding inhibitor of the free enzyme in inducing inactivation. The concentration of zinc that is effective for action is in the micromolar range. The finding that E. coli ornithine transcarbamoylase can be induced to express co-operativity in binding its substrates has recently been confirmed by site-directed mutagenesis experiments. When the active site residue Arg106 is altered to a glycine, the resultant mutant enzyme exhibits both homotropic and heterotropic interactions towards its substrates. In view of the quaternary structure of holoenzyme aspartate transcarbamoylase, the “silent” co-operativity of ornithine transcarbamoylase is of particular interest in the study of evolution of complex, regulatory proteins.
Reconstitution of stellacyanin as a case of direct Cu(I) transfer between yeast copper thionein and 'blue' copper apoprotein
1983, FEBS Letters
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.
Kinetics and mechanisms of the recombination of Zn2+, Co2+, and Ni2+ with the metal-depleted catalytic site of horse liver alcohol dehydrogenase
1983, Journal of Inorganic Biochemistry
The kinetics of the recombination of the metal-depleted active site of horse liver alcohol dehydrogenase (LADH) with metal ions have been studied over a range of pH and temperature. The formation rates were determined optically, by activity measurements, or by using the pH change during metal incorporation with a pH-indicator as monitor. The binding of Zn²⁺, Co²⁺, and Ni²⁺ ions occurs in a two-step process. The first step is a fast equilibrium reaction, characterized by an equilibrium constant K₁. The spectroscopic and catalytic properties of the native or metal-substituted protein are recovered in a slow, monomolecular process with the rate constant k₂. The rate constants k₂ are 5.2·10⁻²sec⁻¹ (Zn²⁺), 1.1·10⁻³sec⁻¹ (Co²⁺), and 2· 10⁻⁴sec⁻¹ (Ni²⁺). The rate constants increase with increasing pH. Using temperature dependence, the activation parameters for the reaction with CO²⁺ and Ni²⁺ were determined. Activation energies of 51 ± 2.5 kJ/mol (0.033 MN-Tris-(hydroxymethyl)methyl-2-aminomethane sulfonic acid (TES), pH 6, 9) for Co²⁺ and 48.5 ± 4 kJ/mol (0.033 M TES, pH 7, 2) for Ni²⁺ at 23°C were found. The correspondent activation entropies are − 146± 10 kJ/mol °K for Co²⁺ and − 163 ± 9 kJ/mol °K for Ni²⁺. Two protons are released during the binding of Zn²⁺ to H₄Zn(n)₂ LADH in the pH range 6.8–8.1. The binding of coenzyme, either reduced or oxidized, prevents completely the incorporation of metal ions, suggesting that the metal ions enter the catalytic site via the coenzyme binding domain and not through the hydrophobic substrate channel.
A kinetic study of the reconstitution of azurin from Cu(II) and the apoprotein
1979, Archives of Biochemistry and Biophysics
The kinetics of reconstitution of Pseudomonas aeruginosa azurin from its apoprotein and copper(II) salts have been studied using absorbance at 625 nm and fluorescence emission at 308 nm as monitors of the process. At low Cu(II) concentrations the rates of both absorbance and fluorescence changes are linearly dependent on Cu(II) concentration. At higher Cu(II) concentrations the rate of absorbance change is independent of Cu(II) concentration. The rates of both absorbance and fluorescence changes as a function of pH suggest that the titration of a single ionizable group is important for the Cu(II)-dependent reaction. Overall analysis of the kinetics suggests that the fluorescence change and the absorbance change are associated with at least two steps in the overall pathway of the formation of the metal-protein complex, and that the copper(II) and tryptophan environments in this protein, though perhaps spatially close, may be distinct.
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1978, Journal of Molecular Structure

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Archives of Biochemistry and Biophysics

Abstract

References (13)

Biochim. Biophys. Acta

J. Biol. Chem

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochemistry

Fed. Eur. Biochem. Soc. Letters

Cited by (14)

Studies on plantacyanin. IV. Reconstitution with Cu-thionein, oxidation by cytochrome oxidase and autooxidation in the presence of cardiolipin

Zn<sup>2+</sup> regulation of ornithine transcarbamoylase. I. Mechanism of action

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

Kinetics and mechanisms of the recombination of Zn<sup>2+</sup>, Co<sup>2+</sup>, and Ni<sup>2+</sup> with the metal-depleted catalytic site of horse liver alcohol dehydrogenase

A kinetic study of the reconstitution of azurin from Cu(II) and the apoprotein

Magnetic resonance studies of some metal - peptide complexes - models of natural systems