pH dependent copper binding properties of a CuA azurin variant with both bridging cysteines replaced with serines

A double mutant of Cu_A azurin was prepared in which both bridging cysteine thiolate ligands of the binuclear Cu_A center were replaced by serine. The copper binding properties of this protein were investigated, and shown to be pH dependent. At lower pH (5.2 ± 0.1), the protein binds one copper per protein molecule as demonstrated by electrospray ionization mass spectrometry. Copper titrations resulted in electronic absorptions at 730 nm (peak) and ca. 330 nm (shoulder) in the UV-Vis spectrum. EPR data show a four line pattern with hyperfine A_∥ = 150 G and g_∥ and g_⊥ values 2.32 and 2.03, characteristic of a type II (T2) copper. Superhyperfines to two nitrogen atoms were also observed. At higher pH (8.5 ± 0.1), the protein binds upto two copper atoms per protein molecule, and copper titrations exhibit a blue transition at 595 nm in the UV-Vis spectrum. The EPR data are consistent with two monomeric sites very similar to one another having hyperfines A_∥ = 182 and 150 G, g_∥ = 2.24 and 2.22 and a similar g_⊥ value of 2.01. These results indicate that both bridging cysteines play a critical role in the Cu_A center, and replacing them with serines is not enough to maintain the symmetrical diamond core structure or the characteristic electronic and functional properties of the Cu_A center.     

The interaction of nitric oxide with ascorbate oxidase.

1. The reaction of nitric oxide with oxidized and reduced ascorbate oxidase (L-ascorbate: oxygen oxidoreductase, EC 1.10.3.3) has been investigated by optical absorption measurements and electron paramagnetic resonance, and the results are compared with those of ceruloplasmin. 2. Upon anaerobic incubation of oxidized ascorbate oxidase with nitric oxide a decrease of the absorbance at 610 nm is found, which is due to an electron transfer from nitric oxide to Type-1 copper. 3. In the presence of nitric oxide the EPR absorbance of ascorbate oxidase decreases and shows predominatly a signal with characteristics of Type-2 copper (g parallel = 2.248; A parallel = 188 G), whereas the type-1 copper signal has vanished. 4. Comparison of the intensities of the EPR signals before and after NO-treatment points to the presence of one Type-2 and three Type-1 copper atoms per molecule of ascorbate oxidase. 5. It is shown that the changes in the optical and the EPR spectrum of ascorbate oxidase induced by nitric oxide are reversible. No difference in enzymic activity is found between the native enzyme and the NO-treated enzyme after removal of nitric oxide.     

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.     

An electron paramagnetic resonance study of native and modified freeze-dried cupric insulin hexamer.

Cupric insulin was modified by the addition of cross-linking disulphide bridges between hexamers. The electron paramagnetic resonance (EPR) spectrum of this freeze-dried material was compared with that of freeze-dried unmodified cupric insulin containing various amounts of copper and added water. The modified insulin was found to have cupric ion sites magnetically very similar to that of native insulin containing two cupric ions per hexamer. Native hexamer produced in the presence of 2 Cu(II) ions per hexamer gave, after freeze-drying, an EPR spectrum with A_∥^Cu=16.5 mT, g_∥=2.285 and g_⊥=2.059 (site 1). The use of 4 or 6 Cu(II) ions per hexamer resulted in spectra with two components-a major component with the same A_∥^Cu and g_∥ values as the sample containing 2 Cu(II) ions (site 1) and an additional minor component (site 2). These sites have been identified with the analogous zinc binding site within the hexamer formed by three B-10 histidine residues (site 1) [1, 2] and the site formed by the B-1 α-amino and A-17 glutamyl-γ-barboxylic acid functions where excess zinc is bound (site 2) [3, 4]. The addition of water to native hexamer containing 2, 4, or 6 Cu(II) ions resulted in the appearance of three distinct EPR absorptions, one of which had the same parameters as the freeze-dried native insulin containing 2 Cu(II) ions per hexamer (site 1). Two further sites appeared (3 and 4) with the following parameters: A_∥^Cu=15.0 mT, g_∥=2.353, and g_⊥=2.07; A_∥^Cu=16.5 mT, g_∥=2.315, and g_⊥=2.07, respectively.     

A spectral investigation of the copper(II) complex of the antitumor compound bleomycin

A thorough spectral investigation of the copper(II) complex of the antitumor compound, bleomycin, has been carried out in solution employing optical, difference optical, electron spin resonance, and circular dichroism techniques. The optical spectrum of a pH = 7 solution of the 1:1 complex between copper(II) and bleomycin is characterized by a broad weak band in the visible region (λ_max = 610 nm) that cannot be resolved and intense ultraviolet bands at 317 (? = 2800), 327 (shoulder), 250 (? = 4700), and 257 nm (shoulder). The circular dichroism spectrum in the visible region shows the broad and weak visible absorption band contains at least three components (558, 675, and 880 nm) that are likely to be “d-d” in origin. The electron spin resonance spectrum is characteristic of a tetragonal d⁹ copper(II) system showing no rhombic distoritions at X-band frequencies (g_x = g_y ± 0.002). The spin Hamiltonian parameters for the pH = 7.0 solution corrected for second order effects are A_∥ = 177 × 10^?4 cm^?1, A_⊥ ? 15 × 10^?4 cm^?1, g_∥ = 2.214, g_⊥ = 2.039. Most interesting was the observation of extra hyperfine splitting due to endogenous nitrogen coordination in a 30% glycerol glass (A^N = 12.0 × 10^?4 cm^?1). That pattern is best interpreted as a seven-line sequence associated with three liganded nitrogens. A dramatic change in all spectral properties occurs when the pH of the copper(II)-bleomycin complex is lowered to 2.5. All these data taken together suggest a CuN₃O coordination complex in solution. Details and justifications as well as a discussion of the limitations of the interpretations are presented.     

The interaction of ascorbate oxidase with L-dopa,L-tyrosine and 3,4-dihydroxycinnamic acid. Evidence for irreversible damage of the enzyme during catechol oxidase activity

The aerobic interaction between ascorbate oxidase and L-tyrosine, L-3,4-dihydroxyphenylalanine or 3,4-dihydroxycinnamic acid in 1:10 molar ratio was followed by optical absorption, CD and EPR spectroscopy in 0.1 M phosphate buffer at pH 5.0. While the spectra of the system ascorbate oxidase—L-tyrosine remain practically unaffected after several hours, indicating that no oxidation of the amino acid occurs in the conditions employed, rather drastic changes can be observed in the spectra of the ascorbate oxidase-catechol systems. In particular, while the optical absorption below 500 nm increases markedly due to the formation of the substrate oxidation products, an irreversible decrease in intensity of the absorption, CD and EPR spectral features associated with the blue copper(II) chromophores indicates that a partial loss of Type 1 copper by ascorbate oxidase has occurred during this secondary catechol oxidase activity. A copper species characterized by weak positive CD activity at 370 nm and EPR signal at intermediate field between those of the Type 2 and Type 1 coppers can be detected in the early stages of the reaction. The irreversible damage undergone by the protein during catechol oxidase activity may have biological significance and accounts for the low yield of purified enzyme obtained when the crude enzyme extract is left in prolonged contact with low molecular weight cell components, rich in σ-diphenolic compounds.     

The metal site of Pseudomonas aeruginosa azurin,revealed by a crystal structure determination of the co(II) derivative and co-EPR spectroscopy

The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 Å resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S^δ atom is increased to 3.3–3.5 Å and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1–2.4 Å. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values g_x′ = 5.23; g_y′ = 3.83; g_z′ = 1.995, and hyperfine splittings A_x′ = 31; A_y′ = 20–30; A_z′ = 53 G) and indicates that the ligand field is close to axial. Proteins 27:385–394, 1997. © 1997 Wiley-Liss, Inc.     

Isolation from bovine liver mitochondria of a soluble ferredoxin active in a reconstituted steroid hydroxylation reaction.

An iron-sulfur protein has been isolated from bovine liver mitochondria and purified 140-fold on DEAE-cellulose and Sephadex G-100. During the isolation the protein was detected by its NADPH-cytochrome $\text{c}$ reductase activity in the presence of adrenal NADPH-ferredoxin reductase. The molecular weight of the protein (12,400), the optical spectrum (peaks at 414 nm and 455 nm which disappear upon reduction), and the EPR spectrum (g_x = g_y = 1.935 and g_z = 2.02) were typical for a ferredoxin. In the presence of soluble adrenal cytochrome P₄₅₀, ferredoxin reductase and NADPH, this protein could support the formation of pregnenolone from cholesterol. Under similar conditions, but in the presence of a cytochrome P₄₅₀ solubilized from rat liver mitochondria, cholesterol was transformed into a more polar compound tentatively identified as 26-hydroxycholesterol.     

An EPR Study of NO Bonding to Erythrocruorin from the Earthworm Octolasium Complanatum

The EPR properties of the nitric oxide derivative of Octolasium complanatum erythrocruorin have been investigated as a function of the concentration of protons and cations which are known to affect the oxygen-linked allosteric equilibrium. The EPR spectrum has a rhombic shape with g_x = 2.08, g_z = 2.005, and g_y = 1.99, and remains unchanged under all the experimental conditions used. A supernyperfine pattern consisting of nine equally spaced lines is present in the g_z region indicating an interaction with two nonequivalent nitrogen atoms, one contributed by the nitric oxide and the other by the proximal histidine. The constancy of the EPR spectrum suggests that changes in the allosteric equilibrium do not involve differences in the strain of the Fe(II)-histidine bond as in tetrameric hemoglobins.     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 °K) and potentiometric titration.Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix “N”) resolves into two single electron titrations with E_{m 7.2} values of ?380±20 mV and ?240±20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g_// = 2.03, g_⊥ = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with g_z = 2.03, g_y = 1.94 and g_x = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g_// = 2.05 and g_⊥ = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high E_{m 7.2} value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with E_{m 7.2} = ? 240±20 mV is defined as Center N-3 (g_z = 2.10, (g_y = 1.93?), g_x = 1.87); the ?405±20 mV component as Center N-4 (g_z = 2.11, (g_y = 1.93?), g_x = 1.88). At temperatures close to 4.2 °K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with E_{m 7.2} = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar E_{m 7.2} values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual E_{m 7.2} values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the E_{m 7.2} of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. E_{m 7.2} values and EPR lineshapes for the other ironsulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show E_{m 7.2} values around -250 mV; Center N-2 exhibits a high E_{m 7.2} (?20±20 mV), while Center N-1a shows a very low E_{m 7.2} (?380±20 mV).     

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.     

On the role of a cuprous ion intermediate in the galactose oxidase reaction

The Cu⁺² electron spin resonance spectrum of galactose oxidase (galactose:O₂ oxidoreductase, E.C. 1.1.3.9) indicates that the metal is in a pseudo-square planar environment. The electron g values are: g_zz = 2.273, g_xx = 2.058 and g_yy = 2.048. The copper nuclear hyperfine constants are (in Gauss): A_zz = 176.5, A_xx = 28.8 and A_yy = 30.1. This spectrum is unaltered in either intensity or g or A values under conditions which cause the inhibition of galactose oxidase by superoxide dismutase. No combination of substrates (galactose and O₂) and oxidant traps (superoxide dismutase and catalase) results in the reduction of the cupric ion resonance. Thus, a Cu⁺¹-enzyme does not appear to be a stable intermediate along this enzyme's reaction path.     

Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins

An extension of a method relating chemical structure to the EPR parameters A_∥ and g_∥ is presented. For complexes having the same atoms of ligation, a decrease in charge of the metal-ligand complex decreases g_∥ and increases A_∥. From this analysis, one concludes that in artificial copper proteins as well as in the naturally occurring nonblue copper proteins copper is ligated to oxygen and nitrogen but not to sulfur. A method is presented for the interpretation of EPR changes that occur with ligand exchange reactions at the Type 2 (nonblue) copper sites such as occur in laccase.     

Interaction of ascorbate oxidase with inorganic anions

From the peelings of cucumber Cucumis sativus and marrow squash Cucurbita pepo var. giramontia highly purified ascorbate oxidase preparations were obtained. Molecular weights, optical and EPR spectra, total copper contents and different type copper contents of the both proteins were similar. The effects of NaN3, KCN, I- and F- on the optical and EPR spectra of the proteins were studied. The incubation of ascorbate oxidase with these anions lead to the partial reduction of the copper. The data obtained indicate that F- is bound to the copper atoms of the type 2, and that N5- modifies surroundings of these copper atoms. The copper atoms of types 1 and 2 in both ascorbate oxidases, unlike fungal laccase, are completely reduced under effect of CN-. The bleaching of ascorbate oxidase, observed in alkaline media involves also increasing of the intensity of the band at 330 nm. The results show that three types of copper in ascorbate oxidase have various sensitivities to the inorganic anions. These data are compared with results observed for another blue copper-containing enzymes, such as laccases and ceruloplasmin.     

The interaction between heme and protein in cytochrome c1

The optical spectrum of reduced bovine cytochrome c₁ at 77 K shows a fine splitting of the β-band, which is indicative of the native conformation of the protein. At room temperature, this conformation is reflected in an absorbance band at 530 nm. The exposure of the heme of ferrocytochrome c₁, investigated by means of solvent-perturbation spectroscopy, appears to be extremely sensitive to temperature and SH reagents bound to the oxidized protein. Addition of combinations of potential ligands to the isolated tryptic heme peptide of cytochrome c₁ reveals that only a mixture of methionine and cysteine (or their equivalents) generates a β-band at 77 K which is identical in shape to that of native cytochrome c₁. In the EPR spectrum of a complex of ferrocytochrome c₁ and nitric oxide at pH 10.5, no hyperfine splitting derived from a second ligated nitrogen atom could be detected. The results indicate that methionine and cysteine are the axial ligands of heme in cytochrome c₁. The EPR spectrum of isolated ferricytochrome c₁ is that of a low-spin heme iron compound with a g_z value of 3.36 and a g_y value of 2.04.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trapping of a reaction intermediate by tetranitromethane during catalysis by ribulose biphosphate carboxylase

Illumination at 230 K of dithionite-reduced particles results in the appearance of an EPR detectable radical 13 G wide with g = 2.0033. This radical is formed in a ratio of 2.28 (±0.5)/P700. Investigation of the time course of formation shows two components are present. One (A₁) has g = 2.0051 and the other (A_og= 2.0024. Reduction of A₁ results in an increase in reaction centre triplet formation, subsequent reduction of A_o results in a decrease of triplet formation to the base level. We propose that these components function sequentially in the transfer of electrons from P700 to the iron—sulphur acceptors.     

Spin trapping endogenous radicals in MC-1010 cells: Evidence for hydroxyl radical and carbon-centered ascorbyl radical adducts

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO₂) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO₂ oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (A_N=15.9 G; A_H=22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO₂ treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe²⁺ (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO₂ oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of A_H4=1.8 G.Abbreviation EPR Electron Paramagnetic Resonance