The identity of a new copper(II) electron paramagnetic resonance signal in cytochrome c oxidase

In reoxidation experiments with cytochrome c oxidase (EC 1.9.3.1) in the presence of both reducing substrate and molecular oxygen, a new EPR signal from Cu²⁺ has been observed. The new signal corresponds to 0.45 Cu per functional unit. It is concluded that the new EPR signal originates from Cu²⁺_B, the copper which is EPR-nondetectable in the resting enzyme.Optical absorption changes in the 500–700 nm region accompanies the decay of the new Cu²⁺ EPR signal.Based on the results in this investigation a catalytic cycle for cytochrome oxidase is proposed.     

Kinetics of acid-catalysed dissociation of lead(II) and copper(II) complexes of ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA)

The acid-catalysed dissociation rate constants for PbEGTA²⁻ and CuEGTA²⁻ complexes (where EGTA is ethylenebis(oxyethylenenitrilo) tetraacetic acid) were measured in acetic acid-acetate buffer medium (pH: 3.0–4.8) and perchloric acid solutions ([H⁺] = 0.05–0.15 M), respectively, at a constant ionic strength of 0.15 (NaClO₄). The rate laws shown by the lead(II) and copper(II) complexes are of the form, Rate = {k_d + k_H[H⁺]}[complex] and Rate = {k_d + k_H²[H⁺]²}[complex], respectively. Enthalpy and entropy of activation for acid-independent and acid-catalysed pathways for both the complexes were obtained by the temperature-dependence studies of resolved rate constants in the 16–45°C range. The rate of dissociation of PbEGTA²⁻ is not enhanced by increasing the concentration of acetate ion in the buffer, and the amount of total electrolyte in the reaction mixture has no pronounced effect on the dissociation rates of their the lead(II) or copper(II) complex. Attempts to study the kinetics of stepwise ligand unwrapping in the binuclear Cu₂EGTA complex were unsuccessful due to the extremely rapid dissociation of this complex to yield mononuclear CuEGTA²⁻.     

Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g_|| = 2.24, A_|| = 18.4 mT and g_⊥ = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g_|| = 2.29, A_|| = 16.1 mT and g_⊥ = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu²⁺ with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu²⁺ and three diamagnetic Cu¹⁺ per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per αβγ-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe³⁺ was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu²⁺-EPR signal.     

The effect of phospholipid depletion on the EPR behavior of cytochrome oxidase

Phospholipids are essential components for electron transport activity of cytochrome oxidase. Recently, we have found that the removal of phospholipids from the oxidase affected the copper and low-spin heme signals, and conceivably other paramagnetic centers as demonstrated by EPR spectroscopy. At 4.2–30 °K, the signal amplitudes and power saturation behaviors were studied at approximately g = 2.0 for the copper signal, and in the neighborhood of g = 3.0 for the low-spin heme signal. After depletion of phospholipids the amplitude of the copper signal decreased 25–30% at 12–30 °K and below 12 °K 40–50% under nonsaturating conditions. The amplitude of the low-spin heme signal decreased 60–70% at 4.2–20 °K. Below 14 °K both signals became more resistant to power saturation, but the copper signal was more readily saturated above this temperature, compared to the oxidase with about 25% lipid. After removal of phospholipids, the spectral features of the copper signal remained essentially the same, but the low-spin heme signal broadened and became very asymmetric to show two signals as revealed by the second harmonic EPR spectra. These findings may explain, at least partially, the wide variations in percentage of EPR detectable copper and heme of cytochrome oxidase reported by different laboratories. Unequivocally, the EPR behavior of cytochrome oxidase is not only affected by the protein moiety, but also by the associated phospholipids of the enzyme.     

Buffer-dependent variations in assay of tyrosine aminotransferase holoenzyme

Homogenization of rat liver in Hepes (N-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid), MOPS (2-[N-morpholino]ethanesulfonic acid), Na phosphate, Pipes (piperazine-N,N′-bis[2-ethanesulfonic acid]), TEA (triethanolamine), TES (N-tris[hydroxymethyl]-methyl-2-aminoethanesulfonic acid), Tricine (N-tris-[hydroxymethyl]methylglycine), or Tris (tris[hydroxymethyl]aminomethane), and subsequent assay for supernatant total and holo tyrosine aminotransferase activity using these buffers yields apparent enzyme concentrations which vary depending upon the buffer composition, the ionic strength, and the fold-dilution of the supernatant. A precipitous decrease in the apparent holoenzyme concentration results from a slight dilution of the supernatant with most of the buffers. Some of the dilution effects may be due to dissociation of pyridoxal phosphate from the apoenzyme or to competition between the buffer and pyridoxal phosphate for association with the enzyme. The percentage of the apparent total enzyme which exists as holoenzyme varies from 3% for supernatant prepared in Na phosphate buffer up to 94% for that prepared in Hepes. Inactivation of total enzyme activity occurs to a similar extent resulting from incubation of liver homogenates prepared with Na phosphate, Hepes, or Pipes. The residual apparent holoenzyme activity observed when assayed in the presence of Na phosphate may be due to reaction of an enzyme other than tyrosine aminotransferase. The data provide a basis for explaining the large variation in reported percentage holoenzyme and should also serve as a warning for other holoenzyme assays which use pyridoxal phosphate as a cofactor.     

The Effects of Different Buffers on the Oxidation of DNA by Thiols and Ferric Iron

Because buffers can act as metal ligands, they can effect several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction. Therefore, these reactions were studied in Hepes and phosphate buffers and unbuffered NaCl. Reduction of Fe³⁺ by dithiothreitol (DTT) and cysteine was observed in either Hepes or NaCl solutions, but not in phosphate buffer. Thiyl radicals were observed in Hepes, but there was much less thiyl radical production in the saline or phosphate solutions. Redox cycling between either DTT or cysteine and Fe³⁺ also resulted in dioxygen consumption in Hepes buffer. Reduction of Fe³⁺ and O₂ resulted in the formation of an oxidant capable of producing 8-hydroxy-2′-deoxyguanosine (8-OHdG) in calf-thymus DNA. The highest levels of 8-OHdG were detected when DTT or cysteine and Fe³⁺ were incubated in Hepes, while much less DNA oxidation was detected when the experiment was done in a saline solution, and almost no DNA oxidation occurred in the phosphate buffer. These results demonstrate that the use of different buffers can greatly affect the ability of thiols to promote iron-dependent oxidations. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 125–132, 1998     

Rapid down regulation of insulin receptors in adipocytes artifact of the incubation buffer

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[¹²⁵I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors $\text{in}\text{vitro}$ is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.     

Purification and Properties of Phage 95-induced Lytic Enzyme of Pseudomonas aeruginosa

A lytic enzyme was isolated from the lysate of Ps. aeruginosa infected with a new strain of bacteriophage, phage 95. The enzyme, LE95, was purified by chromatography in twice on IRC50 column and by gel filtration in twice on Sephadex G–75 column. The molecular weight was estimated as 21,000. The optimal condition for the hydrolysis of acetone-dried cells of Ps. aeruginosa was determined to be following: the optimal pH was between 6.5 and 7.0, the temperature about 70°C and the concentration of phosphate buffer about 5 mm. The enzyme was strongly inhibited by Ag⁺, Hg²⁺, Ni², Fe²⁺ and Cu²⁺ ions. When peptideglycan obtained from Ps. aeruginosa was digested by LE95, free amino groups were liberated without release of reducing sugars. The enzyme was suggested to be amidase or peptidase.     

Purification and partial characterization of an acid phosphatase from the body wall of sea cucumber Stichopus japonicus

A high molecular weight (HMW) acid phosphatase from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of anion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). The enzyme was purified 19.3-fold with a total yield of 1.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 147.9 kDa. The enzyme displayed maximum activity at pH 4.0 and 50 °C with p-nitrophenyl phosphate as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg²⁺, whereas inhibited strongly by Cu²⁺ and Zn²⁺. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The amino acid sequences of three segments of the purified enzyme were analyzed by mass spectroscopy, which did not have any homology with previously described acid phosphatase.     

Differential effects of urea on yeast and bovine copper, zinc superoxide dismutases, in relation to the extent of analogy of primary structure.

Incubation in 8M urea (pH 7.4) inactivated yeast Cu, Zn superoxide dismutase with biphasic first order kinetics (k for the decrease from 100% to 16% activity = 6.5 × 10^?3 min^?1; k for the decrease from 16% to 0.1% activity = 2.5 × 10^?3 min^?1). The inactivation was fully reversible on dilution with or dialysis against urea-free buffer. No inactivation was shown to occur in similar experiments with the bovine Cu, Zn enzyme. EPR spectra recorded immediately after addition of 8M urea showed a more axial line shape and a higher A_″ of the copper signal typical of the native enzyme. In the case of the yeast enzyme, this change was more pronounced and further incubation led to a new type of copper signal, typical of the inactivated enzyme. All EPR changes were reversible. Comparative analysis of the amino acid sequence of the two enzymes showed substantial identity of the protein regions contributing the ligands to the metals and the disulfide bridge. Differential destabilization of active sites by urea should be due to replacements in other protein segments, such as the three C-terminal and some N-terminal residues.     

Temperature-dependent permeability of large unilamellar liposomes

The temperature-dependent drug leakage from liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1, by weight) was studied. Experiments were performed in Hepes buffer and 50% fetal calf serum. Large unilamellar liposomes were formed by the reverse phase evaporation process and extruded through a series of polycarbonate membranes with pore sizes of 0.4, 0.2, 0.1 and 0.08 μm. The release of the water soluble radioisotopes cytosine 1-β-D-[³H]arabinofuranoside and [³H]inulin from the aqueous compartment of these liposomes was measured as a function of time and temperature. Both radioisotopes were released at temperatures near 42°C, the solid-to-liquid-crystalline phase transition temperature of these lipids. The percent drug release decreased as the size of the liposomes was reduced. This effect was more pronounced in Hepes buffer than serum. The release of both radioisotopes was greatest at 40°C in Hepes buffer and at 43°C in 50% fetal calf serum. In addition, the rate of drug release was much faster in serum than in buffer. These results suggest that different drug release processes are occurring in buffer and in serum.     

The effect of phosphate buffer in the range of pH 5.80–8.07 on jack bean urease activity

The effect of phosphate buffer on the activity of jack bean urease was studied in the range of pH 5.80–8.07. The inhibition constants of phosphate buffer were determined by measuring initial reaction rates at each pH for a series of buffer concentrations at a series of urea concentrations. It was shown that: (1) at pH 5.80–7.49 the buffer is a competitive inhibitor of the enzyme with K_i,buffer increasing from 0.54 mM for pH 5.80 to 362 mM for pH 7.49, (2) the values of pK_i,buffer are pH-dependent exhibiting a slope of −1 at pH 5.80–6.5 and a slope of −2 at pH 6.5–7.49, (3) from pH 7.62 as the pH is further raised the competitive inhibition of urease by the buffer was not observed, (4) the true competitive inhibitor of urease is H₂PO₄⁻ ion, and (5) pH 6.5 and 7.6 correspond to the ionization constants of the active site groups of urease responsible for the inhibitory strength of H₂PO₄⁻ ion.     

An EPR signal from the "invisible" copper of cytochrome oxidase.

Sulfide is both an inhibitor and a slow reductant of oxidized cytochrome $\text{c}$ oxidase. When the enzyme is exposed to sulfide for short times (one minute or less) and frozen, the resultant electron paramagnetic resonance (EPR) signals show clearly: low spin heme a, low spin heme a₃, the usual “EPR detectable” Cu²⁺ signal (g_⊥ = 2.17, g_⊥ = 2.03), and a new Cu²⁺ signal superimposed on the same region, with (g_⊥ ～ 2.19, g_⊥ = 2.05). This new signal presumably arises because the antiferromagnetic coupling postulated to exist between the iron atom of heme a₃ and this copper is disrupted when heme a₃ is driven to a low spin state by sulfide. The implications of this result with respect to models of the O₂-binding site and redox geometry of oxidase are briefly discussed.     

Studies on the origin of the near-infrared (800–900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 (‘copper signal’) of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (?170 to ?190°C). For some sets of samples spectra were recorded in the range 500–1100 nm. A particular effort was made to study this correlation with what are called ‘mixed valence’ states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205–215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10–15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700–800 nm region did not appear to be more useful than the 800–900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

Degradation of DMPO Adducts from Hydroxyl and 1-Hydroxyethyl Radicals by Rat Liver Microsomes

Hydroxyl and 1-hydroxyethyl radical adducts of 5, 5-dimethylpyrroline N-oxide (DMPO) were prepared by photolysis, and mechanisms for loss of their EPR signals in rat liver microsomal suspensions were evaluated. Rates of NADPH-dependent EPR signal loss were more rapid in phosphate buffer than in Tris buffer. Addition of superoxide dismutase (SOD) partially protected the adducts when Tris was used as a buffer, but was relatively ineffective in the presence of phosphate. The ferrous iron chelator bathophenanthrolene partially protected the spin adducts in the presence and absence of phosphate, but complete protection was observed when SOD was also added. The spin adducts were unstable in the presence of Fe⁺² and K₃Fe(CN)₆, but Fe⁺³ alone had little effect on the EPR signals. The data are consistent with two mechanisms for microsomal degradation of DMPO spin adducts under these conditions. Microsomes form superoxide in the presence of oxygen and NADPH, which attacks these DMPO spin adducts directly. The spin adducts are also degraded in the presence of Fe⁺², and phosphate stimulates this iron-dependent destruction of DMPO spin adducts.     

An EPR Investigation of Human Methaemoglobin Oxidation by Hydrogen Peroxide: Methods to Quantify all Paramagnetic Species Observed in the Reaction

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglabin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H₂O₂ was added at 3- and 10-fold molar excess to 100 μM metHb in 50 mM phosphate buffer, pH 7.4, 37°C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H₂O₂ the EPR signals (- 196°C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (- 196°C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g° = 2.035 and g° = 2.0053), and the other an isotropic singlet (g = 2.0042, AH = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic Pehaviour of both peroxyl (ROO^*) and nonperoxyl (P^*) free radicals were similar, we concluded that ROO^* radicals are not formed from P^* radicals by addition of O₂. The time courses for both radicals showed a steady state during the time required for H₂O₂ to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 ± 10^-3 s^-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H₂O₂. The formation of the rhombic Fe(III) non-haemcentres with g = 4.35 was found. Their yield was proportional to the H₂O₂ concentration used and the centers were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at —196°C were used.     

The configuration of the Cu2+ binding region in full-length human prion protein

The cellular prion protein (PrP^C) is a Cu²⁺ binding protein connected to the outer cell membrane. The molecular features of the Cu²⁺ binding sites have been investigated and characterized by spectroscopic experiments on PrP^C-derived peptides and the recombinant human full-length PrP^C (hPrP-[23-231]). The hPrP-[23-231] was loaded with ⁶³Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP^C/Cu²⁺-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu²⁺ structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu²⁺ coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP^C. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction of Aplysia brasiliana Myoglobin with Mn2+ and Cu2+ ions

Myoglobin of Aplysia brasiliana (MbApB) has been recently purified and characterized and it was shown that the amino acid content is quite different from other myoglobins. A large number of aromatic residues was observed together with the existence of a unique histidine at the proximal heme position. Because of the numerous differences in the amino acid sequence between MbApB and whale myoglobin, it was interesting to investigate the interaction of metal ions like Cu²⁺ and Mn²⁺ with MbApB. In the present work Cu²⁺ complexes with Met-MbApB were studied and show a pH transition between different forms of coordination as revealed by EPR measurements. At high pH the EPR spectrum shows the coordination of the metal to at least four nitrogens from ϵ-NH₃ lysine residues. At lower pH in the range 6.0–9.0 the copper binding site shows a pK change of some of the residues involved in metal coordination. Addition of one equivalent Cu²⁺ per protein does not alter the iron EPR signal. The manganese ion has one binding site in MbApB and a binding constant K_a = ( 11.5 ± 0.8) 10³M⁻¹. The binding of Cu²⁺ to MbApB is stronger than Mn²⁺, K_a^Cu2+ >K_a^Mn2+.     

Synthesis and Study of Copper-Containing Polymers Based on Sulfated Arabinogalactan

Synthesis of water-soluble copper-containing sulfates of arabinogalactan was carried out for the first time by the ion exchange method. Their composition and structure were studied by the methods of elemental and chemical analysis, X-ray spectral microanalysis, atomic force microscopy (AFM), infrared spectroscopy (FTIR), and electron paramagnetic resonance (EPR). According to the AFM data, the surface of copper-containing polymer films does not have inclusions and consists of homogeneous crystallites of a spherical and slightly elongated shape and transverse dimensions of about 100 nm. The composition of copper- containing polymers was studied by the chemical method and X-ray spectral microanalysis. The absence of nitrogen in the obtained polymer indicates the complete replacement of ammonium cations in the ammonium salt of AG sulfate with the copper cations. The IR spectrum of copper-containing AG sulfate is similar to that of the sodium salt of sulfated arabinogalactan. Superposition of two signals was observed in the EPR spectrum of copper-containing AG sulfate. One of them belongs to isolated Cu²⁺ ions; another, to associated Cu²⁺ ions in the salt-like compounds. The integral intensity of isolated Cu²⁺ ion signals (anisotropic signal) and associated ions (isotropic signal) depends on the copper content in the polymer. Water-soluble coppercontaining polymers of AG sulfates have prospects for their use in medicine.     

A comparative study of antioxidative defense system in the copper and temperature acclimated strains of <Emphasis Type="Italic">Anabaena doliolum</Emphasis>

This study provides first hand comparative account of growth and antioxidative defense system of the wild type, Cu²⁺ and temperature treated wild type and acclimated strains of Anabaena doliolum Bharadwaja against Cu²⁺ and high temperature. The acclimated strains showed perceptible growth at 250 μM Cu²⁺ and 47°C temperatures, respectively. In contrast to this the wild type strain on exposure to 50 μM Cu²⁺ and 47°C temperature depicted almost complete inhibition of growth. However, the peroxide content was significantly higher in the acclimated strains than the wild type. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) showed maximum activity at high temperature followed by Cu²⁺ acclimated and minimum in the wild type strains. The ascorbate (ASC) and glutathione (GSH) contents were increased by 2.3 and 43.3, and 15.5 and 36.5-fold in Cu²⁺ and 47°C acclimated strains, respectively. However, when the wild type strain was subjected to Cu²⁺ and temperature all antioxidative enzymes except SOD showed inhibition of their activity. In case of wild type the GSH content was inhibited by 0.39-fold at 50 μM Cu²⁺ but the ASC content registered increase by 2 and 2.7-fold on subjecting to Cu²⁺ and temperature, respectively. Thus increased activity of enzymatic antioxidants as well as accumulation of ascorbate and glutathione in both the acclimated strains suggests that enzymatic and non-enzymatic antioxidants help in the acclimation of A. doliolum Bharadwaja against Cu²⁺ and high temperature. However, inhibition of antioxidative defense system of wild type under Cu²⁺ and heat stress appears to be the reason for its non survival. In view of the appreciable increase in the level of antioxidants as well as greater inhibition of specific growth rate in temperature than Cu²⁺ acclimated strains, temperature (47°C) is proposed to be is more deleterious to the organism than copper (250 μM).