Optical properties and a new epr signal from type 3 copper in metal-depleted Rhus vernicifera laccase

Due to conflicting reports on the properties of Rhus laccase depleted in type 2 copper a further investigation of this protein derivative has been undertaken. In contrast to most other reports it is shown that the type 3 copper site retains its absorbance at 330 nm when type 2 copper is removed. The type 3 copper ions are oxidized in the resting protein and part of the type 3 Cu(II) can be made electron paramagnetic resonance (epr) detectable on reduction by ascorbate. This new epr signal is highly rhombic and the epr parameters are comparable to those found in other metalloproteins containing Cu(II) in binuclear sites. Certain preparations of type 2 deficient protein exhibit lower extinction coefficients at 330 nm. Since these protein derivatives have lost some type 3 copper, it is inferred that the absorbance at 330 nm is dependent on a native type 3 copper site. Also in contrast to other reports, it is found that the extinction coefficient at 614 nm of the type 1 Cu(II) decreases from 5700 to 4700 M^?1cm^?1 when type 2 copper is removed. The oxidized-reduced difference spectrum also shows a substantial decrease in the absorbance between 700 and 800 nm. The changes in absorbance above 600 nm are probably due to a modification of the type 1 Cu(II) site on removal of type 2 copper. The present results also suggest some explanations to the apparent discrepancies among the earlier reports.     

Studies of the copper and heme cofactors of pseudomonad L-tryptophan-2,3-dioxygenase by electron paramagnetic resonance spectroscopy

l-Tryptophan-2,3-dioxygenase, (EC 1.13.1.12) purified from Pseudomonas acidovorans, is inactivated on aerobic aging or on treatment with K₃Fe(CN)₆, but regains activity in the presence of reducing agents such as sodium ascorbate. Examination of oxidized, inactive enzyme by electron paramagnetic resonance (epr) spectroscopy has revealed the presence of high spin ferriheme (g = 6.2) and of Cu(II) (g_⊥ = 2.065, g_∥ = 2.265) in the enzyme.The epr signal of Cu(II) in inactive tryptophan oxygenase is attenuated on the addition of ascorbate, whereas the high spin ferriheme signal is unaffected, indicating that the site of action of reducing agents in activating the enzyme is the enzymic copper. Quantitation of the Cu(II) signal in inactive tryptophan oxygenase by double integration accounts for 45% of the total copper.Addition of l-tryptophan to either inactive or active enzyme produces a decrease of 44 ± 5% of the epr signal of high spin ferriheme and the emergence of the epr signal of a low spin ferriheme (g_{1, 2, 3} = 2.66, 2.20, 1.81). Disappearance of the high spin ferriheme is hyperbolic (Hill coefficient, n = 1.02) with respect to l-tryptophan concentration, while the appearance of the low spin ferriheme is sigmoidal (Hill coefficient, n = 1.33) with respect to l-tryptophan concentration. The characteristics of the epr signal of this low spin ferriheme are intermediate between those of the signals of the hydroxides of hemoglobin and myoglobin and those in which two histidines are ligated to the ferriheme of hemoglobin. This may be the first example of the observation by epr of an allosteric parameter of an enzyme.     

Spectroscopic and catalytic properties of Rhus vernicifera laccase depleted in type 2 copper.

1. The type 2 copper in Rhus vernicifera laccase was completely removed without loss of other types of copper. The properties of this protein derivative and the role of type 2 copper in the catalytic action of laccase was investigated. 2. The molar extinction coefficient at 614 nm of the blue chromophore decreases from 5700 to 4700 cm-1 on removal of type 2 copper. There are no apparent absorption changes at other wavelengths in the visible or near ultraviolet region when this copper is taken away. The electron-paramagnetic-resonance (epr) parameter A parallel and the linewidth of type 1 Cu2+ decreases on removal of type 2 copper. 3. The rate of reduction of type 1 Cu2+ is not affected by removal of type 2 copper but the reduction of the two-electron acceptor is greatly impaired. These results strongly support the idea that type 1 Cu2+ is the primary site for electron transfer between substrate and enzyme and that the two-electron acceptor in the native enzyme is reduced by simultaneous electron transfer from reduced types 1 and 2 copper. 4. Reoxidation of types 1 and 3 copper and the formation of the oxygen intermediate are the same processes in native and type-2-depleted enzyme. These observations suggests that type 2 copper is not involved in the formation and rapid decay of the oxygen intermediate and that it is not necessary for the stabilization of this intermediate. 5. Two new epr signals are observed on reoxidation of reduced type-2-depleted laccase. One is temporarily formed on re-reduction of reoxidized enzyme and it is suggested that it might arise from copper, possibly type 3 copper. The other one is stable for hours and it is proposed that it might come from a modified oxygen intermediate.     

Copper(II) complexes as superoxide dismutase mimics: Synthesis, characterization, crystal structure and bioactivity of copper(II) complexes

Two new copper(II) complexes of the type [Cu(L)X₂), where L = (E)-N-phenyl-2-[phenyl (pyridine-2-yl)methylene]hydrazinecarboxamide X = Cl/Br have been synthesized and characterized by elemental analyses, FAB (fast atomic bombardment) magnetic measurements, electronic absorption, conductivity measurements cyclic voltammetry (CV) and Electron paramagnetic resonance (epr) spectroscopy. The structures of these complexes determined by single crystal X-ray crystallography show a distorted square based pyramidal (DSBP) geometry around copper(II) metal center. The distorted CuN₂OX (X = Cl/Br) basal plane in them is comprised of two nitrogen and one oxygen atoms of the meridionally coordinated ligand and a chloride or bromide ion and axial position is occupied by other halide ion. The epr spectra of these complexes in frozen solutions of DMSO showed a signal at g ca. 2. The trend in g-value (g_|| > g_⊥ > 2.00) suggest that the unpaired electron on copper(II) has d_x²-y² character. Biological activities in terms of superoxide dismutase (SOD) and antimicrobial properties of copper(II) complexes have also been measured. The superoxide dismutase activity reveals that these two complexes catalyze the fast disproportionation of superoxide in DMSO solution.     

Effect of temperature on ppp(A2''p)nA binding protein activities in rabbit reticulocyte lysates and other mammalian extracts

New epr features consistent with a novel type of Cu(II) are observed in partially reduced Type 2 copper depleted laccase molecules. Cu(II) hyperfine lines appear near 2590 G and 2770 G, and a rhombic g1 feature is also observed. These reflect a Cu(II) emergent on reductive disruption of the binuclear Type 3 site in T2D laccase. Additionally, much of the new, magnetically isolated Cu(II) is retained on full reoxidation of partly reduced Type 2 copper depleted laccase. The proportion of disrupted Type 3 Cu(II) sites remaining after reoxidation appears to depend on the prior distribution of electrons within T2D laccase.     

The binding of copper ions to daunomycin and adriamycin

Using visible absorption, CD, ¹H nmr, and epr spectroscopy, the Cu(II) binging properties of daunomycin, adriamycin, and N-trifluoroacetyl daunomycin in water and ethanol have been explored. The drugs form two water soluble complexes having Cu-drug stoichiometries of 1:1 and 1:2, and with apparent pK_as of formation of 5.6 and 6.5, respectively. At pH values above ～8, the drugs form insoluble polymeric complexes with Cu(II). Similar species are also observed in ethanol. The structure of the compounds have been interpreted in terms of binding of the deprotonated hydroxyquinone portion of the drug to the copper ion. No evidence for the binding of the amino group on daunosamine was found.     

Vanadyl(IV) conalbumin. II. Mixed metal and anion binding studies

Electron paramagnetic resonance (epr) studies demonstrate that at low levels of conalbumin (CA) saturation with Fe³⁺ or VO²⁺, a ph-dependent preference of the metal exists for different protein binding-site configurations,A, B, and C. The vanadyl ion epr spectra of mixed VO²⁺, Fe³⁺-conalbumin in which Fe³⁺ is preferentially bound to the N- or C-terminal binding site are consistent with all three configurations being formed at both metal sites. At high pH the spectra suggest interaction between binding sites. In the absence of HCO₃^?, VO²⁺ is bound almost exclusively in B configuration; a full binding capacity of 2 VO²⁺ per CA is retained. Stoichiometric amounts of HCO₃^? convert the epr spectrum from B to an A, B, C type. Addition of oxalate to bicarbonate-free preparations converts the B spectrum to an A′, B, C′ type where the B resonances have lost intensity to the A′ and C′ resonances but have not changed position. The data suggest that configuration B is anion independent and that only one equivalent of binding sites at pH 9 responds to the presence of HCO₃^1? or oxalate by changing configuration but not metal binding capability. The form of the bound anion may be HCO₃^? rather than CO₃^2?. The formation rate of the colored ferric conalbumin complex by oxidizing Fe²⁺ to Fe³⁺ in limited HCO₃^? at pH 9 is also consistent with one equivalent of sites having different anion requirements than the remaining sites. Increased NaCl or NaClO₄ concentration or substitution of D₂O for water as solvent affect the environment of bound VO²⁺, but the mechanisms of action are unknown.     

Removal of type 2 Cu from ascorbate oxidase and laccase by reaction with n,n-diethyldithiocarbamate

The type 2 Cu of ascorbate oxidase from zucchini peelings can be rapidly removed by reaction with a tenfold excess N,N-diethyldithiocarbamate (DDC) in air, while other chelating agents, such as EDTA, require anaerobic reducing conditions. The type 2 Cu of laccase from Rhus vernicifera is never removed under aerobic conditions. In anaerobiosis and in the presence of a reducing agent, EDTA is also unable to remove the copper unless a smaller lipophilic molecule (DDC or dimethylglyoxime) is present, acting as a mediator. Type 1 Cu is not involved in the reaction of ascorbate oxidase with DDC, but reduction of type 3 Cu is probably required for type 2 Cu depletion, suggesting interdependence of type 2 and type 3 copper. Type 2 Cu is less exposed in laccase, possibly because of the large carbohydrate content of this protein.     

Azide binding to the trinuclear copper center in laccase and ascorbate oxidase.

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1:1 to 1:7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g approximately 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g approximately 1.97 signal, with similar saturation characteristics was found in the AO : azide (1:7) sample. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti-ferromagnetic coupling of the type 3 (T3) Cu(II) pair. Analysis of the position of the g < 2 signals suggests that the distance between the dipolar coupled Cu(II) pair is shorter in laccase than in AO. The proximity of T2 Cu(II) to the S = 1 Cu(II) pair enhances its relaxation rate, reducing its signal intensity relative to that of native protein. The disruption of the T3 anti-ferromagnetic coupling occurs only in part of the protein molecules, and in the remaining part a different azide binding mode is observed. The 130 K EPR spectra of AO and laccase with azide (1:7) exhibit, in addition to an unperturbed T2 Cu(II) signal, new features in the g parallel region that are attributed to a perturbed T2 in protein molecules where the anti-ferromagnetic coupling of T3 has not been disrupted. While these features are also apparent in the AO : azide sample at 10 K, they are absent in the EPR spectra of the laccase : azide sample measured in the range of 6-90 K. Moreover, pulsed ENDOR measurements carried out at 4.2 K on the latter exhibited only a reduction in the intensity of the 20 MHz peak of the 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear : azide complex.     

Versatile nuclearity in copper complexes with ortho functionalized 1,3-bis(aryl)triazenido ligands

The synthesis, characterization and crystal structures of three new copper complexes derived from 1,3-bis(aryl)triazenido ligands bearing either a methoxycarbonyl, methylthio or a hydroxymethyl group in the ortho position of one of the aromatic rings are reported. In addition to the coordination of the triazenido fragment, the Lewis basic groups coordinate to the copper centers to form complexes with different nuclearity: {1-[2-(methoxycarbonyl)phenyl]-3-[4-methylphenyl]}triazene and {1-[2-(methylthio)phenyl]-3-[4-methylphenyl]}triazene form stable dinuclear and tetranuclear Cu(I) complexes, respectively. Reaction of {1-[2-(hydroxymethyl)phenyl]-3-[4-methylphenyl]}triazene with either Cu(I) or Cu(II) results in a novel Cu(II) hexanuclear macrocyclic complex.     

Structural, magnetic and spectroscopic characterization of two unusual end-on bis(μ-acetato/μ-nitrato) bridged copper(II) complexes with N′-[phenyl(pyridin-2-yl)methylidene]furan-2-carbohydrazide and (2E,4Z)-N,2-dimethylhepta-2,4,6-trienamide-1-phenyl-1-pyridin-2-ylmethanimine (1:1) as capping ligands

Two new binuclear end-on bis(μ-acetato/μ-nitrato) bridged complexes with two NNO donor ligands, viz., [(L¹)Cu(μ-CH₃COO)₂Cu(L¹)]·4,4-bipy 1 and [(L²)Cu(μ-NO₃)₂Cu(L²)] 2 where [L¹ = N′-[phenyl(pyridin-2-yl)methylidene]furan-2-carbohydrazide, L² = (2E,4Z)-N,2-dimethylhepta-2,4,6-trienamide-1-phenyl-1-pyridin-2-ylmethanimine (1:1) μ-CH₃COO⁻ = μ-acetato and μ-NO₃⁻ = μ-nitrato) have been prepared and physiochemically characterized. These complexes are structurally characterized by X-ray crystallography. In both complexes, the two copper centers are linked by two acetate or two nitrate groups in end-on bonding fashion. The copper-copper separation is 3.279 Å for 1 and 3.459 Å for 2. The copper ions are pentacoordinated in both complexes. The coordination geometry may be described as close to square pyramidal (SP) stereochemistry with slight distortion to trigonal bipyramidal (TBP) stereochemistry. The polycrystalline epr spectra of these two complexes exhibit the properties commensurate with S = 1 systems. The magnetic moment (μ_eff) for these complexes are below the theoretical value suggesting antiferromagnetic exchange between the copper(II) ions. The cyclic voltammograms (CV) of the two complexes have been investigated. Superoxide dismutase (SOD) activity of these complexes has also been measured. These complexes can catalyze the dismutation of superoxide.     

Some properties of the redox components of cytochrome c oxidase and their interactions.

The electron paramagnetic resonance (epr) properties of cytochrome c oxidase have been examined with special attention to the effect of added ligands and of interactions between the redox components. The fully oxidized preparations have a very small g6 signal which increases greatly as the redox potential is made more negative, a process exactly paralleling the disappearance of the g3 signal. The potential for half appearance or disappearance (E_m), respectively, is 380 mV at pH 7.0 and 300 mV at pH 8.5. This identifies the changes as accompanying reduction of cytochrome a₃ because the E_m of the “invisible copper” is 340 mV and pH independent. Nitric oxide (NO) binds reduced cytochrome a₃ to form a paramagnetic species. This resulting epr signal is strongly dependent on the redox state of cytochrome a, another expression of heme-heme interaction in cytochrome oxidase. The NO compound is also unique in that under the appropriate conditions three of the four redox components (cytochrome a₃, cytochrome a, and the “visible” copper) are epr active. In potentiometric titrations in the presence of azide the formation of the azide compound responsible for the g2.9 signal appears to require reduction of both cytochrome a₃ and the “invisible copper.” An internal sulfur compound is present which, at alkaline pH values, can bind the heme responsible for the g6 signal and change it to a low-spin sulfur compound with a signal at approximately g2.6. Evidence is also presented for the cytochrome c oxidase in situ being an equilibrium mixture of two different conformational states.     

Vanadyl(IV) conalbumin. I. Metal binding site configurations

Electron paramagnetic resonance (epr) and ultraviolet difference spectroscopy of vanadyl conalbumin indicate a binding capacity of two vanadyl ions, VO²⁺, per protein molecule in the pH 8–11 range; the binding capacity drops in the pH 6–8 range with an apparent pK_a′ = 6.6. Iron-saturated conalbumin does not bind vanadyl ions, which suggests common binding sites for iron and vanadium. Ultraviolet difference spectroscopy indicates 2–3 tyrosines are involved in the binding of each metal ion; pH titrations show that three protons are released per vanadyl ion bound by conalbumin. Room and liquid nitrogen temperature X-band (ca. 9.2–9.5 gHz) epr spectra show that the vanadyl ion binds in three magnetically distinct environments (A, B, and C) that arise from interconvertible metal site configurations. These configurations are probably examples of conformational substrates of the protein. Q-band (ca 34 gHz) epr spectra resolve the spectral features more clearly and show that two configurations (A and B) have axially symmetric epr parameters but angles of noncoincidence of 12° and 8°, respectively, between the z components of the g and nuclear hyperfine tensors. The third (C) configuration has rhombic magnetic symmetry and a 6° angle of noncoincidence. These observations demonstrate that the metal sites are of low symmetry and are flexible in their geometry about the metal.The isotropic g and nuclear hyperfine tensor values and the line widths used in computer-simulated epr spectra are consistent with four oxygen or three oxygen and one nitrogen donor atoms binding equatorially to the VO²⁺ group. The apparent stability constant indicates that vanadyl ion binds to conalbumin approximately twelve orders of magnitude more weakly than iron to human serotransferrin but still sufficiently strongly to overcome hydrolysis.     

The formation constants and structures of copper(II)-glycinehydroxamic acid complexes

Formation constants for the complexes formed between copper(II) and glycinehydroxamic acid (H₂NCH₂CONHOH) have been measured potentiometrically at 25.0 °C and I = 0.10 M (NaClO₄). The existence of a dimeric copper(II) species has been shown from the line broadening of the epr spectrum of a solution at room temperature. The structures of the complexes are discussed and conclusions are drawn based on absorption and epr spectra.     

Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g ～ 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undetectable components reduced appears to be inversely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during the titration. At pH 9.3 the quantity of heme represented in the high-spin signals is < 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

Heme-copper and heme-heme interactions in the cytochrome bo-containing quinol oxidase of Escherichia coli

The cytochrome bo quinol oxidase of Escherichia coli is one of two respiratory O2 reductases which the bacterium synthesizes. The enzyme complex contains copper and 2 mol of b-type heme. Electron paramagnetic resonance (epr) spectroscopy of membranes from a strain having amplified levels of this enzyme complex reveals signals from low- and high-spin b-type hemes, but the copper, now established as a component of the oxidase, is not directly detectable by epr. The high-spin signal from the cytochrome bo complex, which we attribute to cytochrome o, when titrated potentiometrically, gives a bell-shaped curve. The low potential side of this curve is biphasic (Em7 approximately 180 and 280 mV) and corresponds to the reduction/oxidation of the cytochrome(s). The high potential side of the bell-shaped curve is monophasic (Em7 approximately 370 mV) and is proposed to be due to reduction/oxidation of a copper center which, when in the Cu(II) form, is tightly spin-coupled to a heme, probably cytochrome o, resulting in a net even spin system and loss of the epr spectrum. The low-spin cytochrome b titrates biphasically with Em7 values of approximately 180 and 280 mV, similar to the high-spin component but without the loss of signal at high potentials.     

The use of a maleimido spin label to detect conformational changes in cytochrome c oxidase.

Spin labeling with a maleimido spin label has been used to investigate conformational changes of bovine cytochrome c oxidase. These experiments show that the spin label is immobilized to a lesser degree when the enzyme is in the “oxygenated” form than it is in the oxidized state and support the view that the oxygenated form is a conformational variant. Experiments in which the maleimido spin-labeled cytochrome c oxidase was titrated with H₂O₂ reveal that the peroxide-treated enzyme, although possessing an absorption spectrum similar to that of the oxygenated form, has an electron paramagnetic resonance (epr) spectrum that is different from that of either the oxygenated form or the oxidized state. Extremes of pH cause a marked decrease in the degree of immobilization of maleimido spin labels bound to the oxidase. Alterations in the epr spectrum are reversible if the pH is held between 5.3 and 10.2 but are irreversible outside that range. Urea and guanidine hydrochloride also decrease the immobilization of the spin labels bound to the oxidase. The nature of the epr spectra indicates that under these conditions the enzyme assumes a more open conformation. Exposure to concentrations of sodium dodecyl sulfate as high as 10% does not result in as much loss of the immobilization as with urea or guanidine. Detergents such as cholate, Tween 80, and Triton X-100 have no significant effect on the epr spectrum of maleimido spin-labeled cytochrome c oxidase.     

Synthesis, spectroscopic and structural characterization of some novel adducts of copper(II) salts with unidentate nitrogen bases

Syntheses, spectroscopic characterization and single crystal X-ray studies are reported for a number of complexes of copper(II) salts with simple monodentate nitrogen bases. The 1:4 adduct of copper(II) sulfate with 3,5-dimethylpyridine (m₂py) CuSO₄·4m₂py, takes the form [(O₃SO)Cu(m₂py)₄], the Cu-O vector of the square-pyramidal coordination environment being disposed on the 4-axis in tetragonal space group P4/n. The complex CuCO₃·Cu(NCS)₂·4py is a linear polymer, taking the form ?O·Cu(py)₂·O·C{O·Cu(py)₂(NCS)₂}·O·Cu(py)₂? (etc.), all atoms lying in the mirror plane of space group Pnma, excepting the pair of ‘py’ (pyridine) ligands disposed to either side. In Cu(OH)I·3/4I₂·2py·1/2MeCN ≡ [{(py)₂Cu(OH)}₄](I₃)₃I·2MeCN a novel cubanoid tetranuclear cation is found (2-symmetry). The EPR spectra of the above compounds show a trend in the anisotropy of the g-values that correlates well with the crystal structures. Obtained only in small quantities but supported by single crystal X-ray studies are the adduct of Cu(OH)Cl with pyrrolidine (pyrr), Cu(OH)Cl:pyrr (1:3), which takes the centrosymmetric binuclear form [(pyrr)₃Cu(μ-OH)₂Cu(pyrr)₃]Cl₂, the copper atom being disposed in a distorted trigonal bipyramidal array, and the adduct 3CuCl₂·CuO·4quin, [Cu₄Cl₆O(quin)₄]Cl₂, which contains the familiar Cu₄Cl₆O core with monodentate quinuclidine (quin) attached to the copper atoms; this compound crystallizes in the cubic space group .     

Effect of CuSO4 and Cu(II)(Gly)2 on some indirect assays for superoxide dismutase activity

The effect of CuSO₄ and Cu(II)(Gly)₂ has been compared with that of superoxide dismutase on the ferricytochrome c reduction and on the nitroblue tetrazolium reduction by an enzymic or chemical flux of superoxide anion radicals as well as on o-dianisidine photooxidation. Both CuSO₄ and Cu(II)(Gly)₂ have been found to inhibit ferricytochrome c reduction as well as the aerobic and anaerobic nitroblue tetrazolium reduction with approximately equal efficiency. Unlike superoxide dismutase they proved capable of inhibiting o-dianisdine photooxidation. The effect of copper either as CuSO₄ or as Cu(II)(Cly)₂ has been established as being due to its interference with the indirect assays for superoxide dismutase activity used. The reasons for this interference have been examined and it is concluded that copper can react with a component of the indirect assay system and depending on the method used it either mimics SOD or acts contrary to the enzyme.