Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site

Vanderdeen, H; Hoving, Henk

doi:10.1021/bi00635a004

Cited by 33 publications

(14 citation statements)

References 31 publications

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“…the binding with ansyl during the removal of copper by CN-. This reaction is known ( [28] and Salvato unpublished) : the two copper atoms in the active site are kinetically different [29,30] and are removed sequentially from the protein. When followed by fluorescence, the kinetics show a typical curve which reflects the different quantum yields of the intermediary products.…”

Section: Discussionmentioning

confidence: 99%

The Binding of 1‐Anilino‐8‐naphthalene Sulfonate to the Hemocyanin of Octopus vulgaris

Ricchelli¹,

Salvato²

1979

European Journal of Biochemistry

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The binding of 1‐anilino‐8‐naphthalene sulfonate (ansyl) to native and copper‐free hemocyanin of Octopus vulgaris has been studied in different conditions by measuring the fluorescence properties of the probe in the presence of hemocyanin. Native hemocyanin, either in the oxygenated or in the deoxygenated state, does not bind ansyl. The binding of ansyl with apohemocyanin induces a strong increase (from 0.004 to 0.6–0.7) of the quantum yield and a blue shift from 520 nm to 460 nm of the emission maximum indicating the presence of ansyl binding sites in the protein. Experimental evidence is reported that the binding occurs at the copper‐binding site of the protein. The dissociation constants of the ansyl‐hemocyanin complexes are equal to about 10−4 M, i.e. they are of the same order of those obtained with other proteins. The number of binding sites (n) of apohemocyanin for ansyl depends on the conformational state of the protein and ranges from 0.15–0.80 mol/mol protein (Mr 500000), depending on pH. ionic strength, and urea concentration. A negative interaction between the ansyl binding sites has been suggested.

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Section: Discussionmentioning

confidence: 99%

The Binding of 1‐Anilino‐8‐naphthalene Sulfonate to the Hemocyanin of Octopus vulgaris

Ricchelli¹,

Salvato²

1979

European Journal of Biochemistry

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“…Dimer hemocyanin Dimer hemocyanin contains a binuclear cupric EPR-detectable active site [Cu(II)Cu(II)]. Its preparation and EPR spectral properties have been investigated and reported (15,16). Addition of a 50-fold excess of N3-to dimer produces a new binuclear cupric derivative, dimer-N3-, as indicated by the changes in the EPR and optical spectra.…”

Section: Methodsmentioning

confidence: 99%

Geometric and electronic structure of oxyhemocyanin: spectral and chemical correlations to met apo, half met, met, and dimer active sites.

Eickman¹,

Himmelwright²,

Solomon³

1979

Proc. Natl. Acad. Sci. U.S.A.

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The chemical and spectral properties of a series of hemocyanin derivatives were systematically compared to provide insight into the geometric and electronic structure of the oxyhemocyanin active site. The binuclear copper site is characterized as two tetragonal Cu(II) Oxygen binds as peroxide (1) to the active site in oxyhemocyanin (2) in the ratio of 1 02:2 Cu and it is generally agreed that the two coppers are in the +2 oxidation state, although a Cu(I)Cu(III) active site has recently been proposed (3). The lack of an electron paramagnetic resonance (EPR) signal for oxyhemocyanin, then, requires the existence of antiferromagnetic coupling or a metal-metal bond between the cupric ions. Magnetic susceptibility studies (4) failed to detect any deviation from Curie behavior, placing only a lower limit of 550 cm-1 on any antiferromagnetic etchange interaction. The optical spectrum also exhibits unique spectral features. The dominant absorption at 345 nm has been variously assigned as a simultaneous pair excitation (SPE) (5) or a ligand-to-metal charge transfer transition (LMCT) with the ligand suggested to be S-(cysteine) (6), O22-(7), or N (histidine) (1). The 570-nm band has been assigned as peroxide to copper charge transfer (1) or d-d transitions (6).We have undertaken a systematic study of a series of hemocyanin derivatives in order of increasing complexity to provide insight into the geometric and electronic structure of the active site of oxyhemocyanin (Fig. 1). The resulting spectral perturbations allow a determination of the oxidation state of the copper ions in each derivative, the existence of both exogenous and endogenous bridges between the two coppers, the coordination mode of peroxide in oxy, and a spectral assignment for the unique absorption features exhibited by oxyhemocyanin. MATERIALS AND METHODSBusycon canaliculatum hemocyanin was purified by ultracentrifugation. A Varian E-9 EPR spectrometer (X-band) was used on frozen solution samples. Low-temperature optical spectra were taken of buffered sucrose solutions with a Cary 17 absorption spectrometer and a Spectrim II cryocooler. Difference spectra of the 300-to 500-nm region were taken with the appropriate protein derivative in the reference beam-e.g., met apo-aquo provided the baseline for met apo-N3-. This was necessary to compensate for residual oxyhemocyanin and the steep slope of the protein absorption in the 300-to 500-nm region. RESULTS AND DISCUSSIONMet apo hemocyanin Met apo hemocyanin is prepared (8) The EPR spectrum of met apo-N3-is given in Fig. 2

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“…Thus the broad signal of binuclear Cu(I1) observed at g = 2 [3] was shown to be obtained when some dioxygen was present in the reaction mixture [4]. Nitrite in the presence of ascorbate yielded mainly a mononuclear Cu(I1) EPR signal at g = 2 [3,4].…”

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confidence: 87%

“…Treatment with NO has to be carried out strictly anaerobically, as in a fast reaction with molecular oxygen NO yields N02, which in aqueous solution dismutates to nitrite and nitrate with the liberation of hydrogen ions. Thus the broad signal of binuclear Cu(I1) observed at g = 2 [3] was shown to be obtained when some dioxygen was present in the reaction mixture [4]. Nitrite in the presence of ascorbate yielded mainly a mononuclear Cu(I1) EPR signal at g = 2 [3,4].The possibilities provided by the Cu(I1) pair, present in H. pomatia methaemocyanin as indicated by the EPR signal at half field [5], encouraged us to reinvestigate the reaction of nitrite and NO with methaemocyanin and with deoxyhaemocyanin in order to elucidate the reaction steps.…”

mentioning

confidence: 99%

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confidence: 99%

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The Reaction of Nitrogen Monoxide and of Nitrite with Deoxyhaemocyanin and Methaemocyanin of Helix pomatia

Verplaetse

Tornout²,

Defreyn

et al. 1979

European Journal of Biochemistry

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Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and NzO in a fast reaction.In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(I1) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra.Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.A green and stable nitrosylhaemocyanin has already been obtained in 1919 on treating anaerobically the deoxyhaemocyanin of Helix pomatia with nitrogen monoxide in slightly acid or in alkaline medium [I]. The addition of NO to deoxyhaemocyanin of Cancer magister gave a similar green derivative which showed a Cu(I1) electron paramagnetic resonance (EPR) signal [2]. The action of NO on the haemocyanin of H.pomatia at pH 5.7 yielded EPR signals of mononuclear Cu(I1) at g = 2 and binuclear Cu(I1) at g = 4 and at g = 2 [3]. Treatment with NO has to be carried out strictly anaerobically, as in a fast reaction with molecular oxygen NO yields N02, which in aqueous solution dismutates to nitrite and nitrate with the liberation of hydrogen ions. Thus the broad signal of binuclear Cu(I1) observed at g = 2 [3] was shown to be obtained when some dioxygen was present in the reaction mixture [4]. Nitrite in the presence of ascorbate yielded mainly a mononuclear Cu(I1) EPR signal at g = 2 [3,4].The possibilities provided by the Cu(I1) pair, present in H. pomatia methaemocyanin as indicated by the EPR signal at half field [5], encouraged us to reinvestigate the reaction of nitrite and NO with methaemocyanin and with deoxyhaemocyanin in order to elucidate the reaction steps. It will be shown that deoxyhaemocyanin yields methaemocyanin in a fast reaction with NO, as indicated by the appearance of an EPR signal at g = 4, as in [ 5 ] , and by the liberation Ahhrrviurions. CD, circular dichroism; EPR, electron paramagnetic resonance.of N 2 0 . Moreover, the presence of NO in the nitrosyl derivative of methaemocyanin will be demonstrated. MATERIALS AND METHODSH. pomatia haemocyanin was prepared by preparative ultracentrifugation of the haemolymph in a Spinco model L, rotor 30, for 2 h at 27500 rev./min at 4°C. The haemocyanin was dissolved in 0.1 M sodium acetate buffer, pH 5.0, and kept under carbon monoxide.Methaemocyanin was obtained by treatment with 25 mM sodium azide or 100 mM potassium fluoride at pH 5.0 and 37 "C for 2 days [6]. The solution was exhaustively dialysed for one week against several changes of 0.1 M acetate buffer, pH 5.0, in Visking dialysis membranes, especially treated [7] in order to remove traces of reducing agents able to regenerate haemocyanin. When needed, the haemocyanin solutions were concentrated by preparative ultracentrifugation in a Spinco...

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Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site

Cited by 33 publications

References 31 publications

The Binding of 1‐Anilino‐8‐naphthalene Sulfonate to the Hemocyanin of Octopus vulgaris

The Binding of 1‐Anilino‐8‐naphthalene Sulfonate to the Hemocyanin of Octopus vulgaris

Geometric and electronic structure of oxyhemocyanin: spectral and chemical correlations to met apo, half met, met, and dimer active sites.

The Reaction of Nitrogen Monoxide and of Nitrite with Deoxyhaemocyanin and Methaemocyanin of Helix pomatia

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