EXAFS studies of binuclear copper site of oxy-, deoxy-, and metaquo-, metfluoro-, and metazidohemocyanin from arthropods and molluscs

Woolery, G. L.; Powers, Linda S.; Winkler, Moritz; Solomon, Edward I.; Spiro, Thomas G.

doi:10.1021/ja00313a019

Cited by 107 publications

(65 citation statements)

References 2 publications

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“…Studies on dinuclear copper model complexes show that even a single hydroxy bridge can establish such a strong coupling between two Cu(I1) centers provided the Cu-0-Cu angle is large (Coughlin and Lippard, 1981;Burk et al, 1981). The interpretation of met-hemocyanin as a bis-hydroxoadduct of the dinuclear site is consistent with the EXAFS data reported by Woolery et al (1984) showing that copper has the same coordination number in met-hemocyanin as well as in oxyhemocyanin. Furthermore, the net charge of the copper complex is not affected in met-hemocyanin by the presence of two hydroxide anions with respect to oxy-hemocyanin binding a perox- ide dianion.…”

Section: Oxy-hcsupporting

confidence: 79%

“…Thus, the axial positions of copper ions are occupied by imidazole nitrogens and no bridging ligand in addition to the p-$:q2 peroxide is present in the oxy-hemocyanin. Furthermore, there is no difference in metal coordination number on going from oxy-hemocyanin to met-hemocyanin, as deduced from the fitting of the first-shell extended X-ray absorption fine structure (EXAFS) data (Woolery et al, 1984).…”

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confidence: 96%

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The Oxidation of Hemocyanin

Beltramini

Bubacco

Casella

et al. 1995

European Journal of Biochemistry

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The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemocyanin has been reinvestigated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. Our study suggests that the protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo-dicopper(I1) complex to which the exogenous anion is also bound. This intermediate in not accumulated and precedes the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met-hemocyanin form. The structure of this dinuclear copper(I1) derivative contains a di-k-hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met-hemocyanin with low affinity ( K = 30 M-I) at pH 7.0, it binds with much higher affinity at pH 5.5 ( K = 1500 M-'), where a second azide ligand also binds in the terminal mode ( K = 20 M-'). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.Keywords: hemocyanin ; copper active site; oxidation; azide binding ; circular dichroism.Hemocyanins are copper proteins found in the hemolymph of several invertebrate species belonging to the phyla of Mollusca and Arthropoda, where they have an oxygen transport and/ or storage function. The biological role of this family of proteins is based on the reversible binding of dioxygen to a dinuclear copper active site. The protein exists in two functional forms, deoxy-hemocyanin and oxy-hemocyanin, their relative abundance depending on oxygen concentration.The deoxy-hemocyanin form contains a dinuclear Cu(1) site. X-ray crystallography data on two deoxy forms, of the lobster Panulirus interruptus hemocyanin (Volbeda and Hol, 1989) and of the horseshoe crab Limulus polyphemus subunit I1 hemocyanin(Hazes et al., 1993; Magnus et al., 1994) are now available.In both proteins each copper ion is bound to three histidine residues. The active-site structure of Panulirus deoxy-hemocyanin consists of a coplanar system of four imidazole nitrogens together with the two metal ions (Cu,, Cu,); the Cu-N and the Dioxygen is bound as a bridge between the two copper ions in a p-q2:q2 mode (Magnus et al., , 1994. This coordination mode is substantiated by a study on a dinuclear copper model complex where pyrazolyl groups replace the imidazole donors, and peroxide is bound as it is proposed for oxy-hemocyanin (Kitajima et al., 1992, Kitajima and Moro-oka, 1994); the absorption properties of this model ...

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Section: Oxy-hcsupporting

confidence: 79%

mentioning

confidence: 96%

The Oxidation of Hemocyanin

Beltramini

Bubacco

Casella

et al. 1995

European Journal of Biochemistry

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“…The set-up on station 8.1 [18], which is equipped with a bent crystal monochromator to correct for vertical divergence of the X-ray beam from the storage ring, and a second crystal [20] for a molluscan hemocyanin, and essentially the same edge spectra have been reported for several other hemocyanins [21], from arthropods as well as from molluscs. Linear combinations of (a) and (c) are used to determine the Cu(I)/Cu(II) ratio in hemocyanin crystals.…”

Section: Cu-k-edge X a Smentioning

confidence: 93%

“…3). In this procedure the assumption was made that the XAS spectra of methemocyanin and oxyhemocyanin are similar, as reported by Woolerey et al [21]. Because the absorption spectra may depend on the crystal orientation with respect to the beam polarization [22], for most crystals or clusters measurements were repeated in different orientations.…”

Section: Cu-k-edge X a Smentioning

confidence: 98%

Spectroscopic investigations of Panulirus interruptus hemocyanin in the crystalline state

Volbeda

Feiters

Vincent

et al. 1989

European Journal of Biochemistry

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Single-crystal ultraviolet spectroscopy, X-ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa.Three types of crystals have been investigated. Type-I monoclinic crystals, which have been used for the X-ray structure determination, contain virtually only deoxyhemocyanin. Type-I1 monoclinic crystals, which are less well ordered than the type-I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type-I1 monoclinic form, subject to gradual conversion to methemocyanin.Hemocyanins are the dioxygen-transporting copper-containing proteins occurring freely dissolved in a large number of invertebrate species. They belong to the class of type-I11 copper proteins, containing a dinuclear copper site. The architecture of the molluscan hemocyanins, large cylinders with molecular mass of up to 10 MDa, differs completely from that of the arthropodan hemocyanins, which are hexamers or multihexamers with molecular mass ranging from 450 kDa to a few million daltons, all built from subunits of z 75 kDa [l -81. In spite of these differences in architecture a wealth of spectroscopic evidence [I, 21 and recent amino acid sequence information [3 -101 indicate that at least half of the dinuclear copper site in the two classes of hemocyanins has similar features.So far, the three-dimensional structure of only one hemocyanin has been solved at atomic resolution by X-ray diffraction techniques: the single-hexameric hemocyanin from the spiny lobster Panulirus interruptus, an arthropod [ll, 121. Using this structure, good progress has recently been made with the structural elucidation of subunit I1 from Limulus polyphemus hemocyanin (Magnus and Lattman, personal communication), but no atomic details have been reported as yet. Amino acid sequence information is now available for ten hemocyanin subunits from six different arthropods. Sequence comparisons show that the Panulirus subunit and its hexamer form the building block of all arthropodan hemocyanins [3 -81. For a meaningful analysis and interpretation of the X-ray structure of P. interruptus hemocyanin with respect to its Abbreviations. XAS, X-ray absorption spectroscopy in the fluorescence mode; Me, SO, dimethylsulfoxide. function, it is very desirable to know the oxidation and oxygenation state of the copper atoms in the crystals used for the structure determination.Spectroscopic studies in solutions have shown that deoxyhemocyanin, which is colourless, contains a pair of Cu(1) ions, while in oxyhemocyanin, which is blue, a pair of Cu(I1) ions is observed [I, 21. In addition, a colourless met form exists containing CU(II)~ without bound oxygen [l, 21. Furthermore, semi-met forms with a Cu(I)/C...

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“…However, several physico-chemical properties of Hc are remarkably similar to those of another copper protein, tyrosinase (Ty; monophenol,o-diphenol:oxygen oxidoreductase, EC 1.14.18.1), widely distributed in animals and plants where it catalyses the o-hydroxylation of monophenols and the O2-dependent oxidation of o-diphenols to quinones (monophenolase and diphenolase activities, respectively) [1,2]. In both proteins each metal ion is bound by three imidazole ligands to yield a dinuclear site having approximately the same metal-metal distance of about 3.6 A [3,4]. Comparative studies on the interaction of Hc and Ty with exogenous molecules showed that the active site of both proteins reacts in the same way with respect to exogenous species: like deoxy-Hc, deoxy-Ty binds reversibly molecular dioxygen at the di-cuprous site to yield the reactive form of the enzyme, oxy-Ty, containing a di-cupric peroxide adduct [1,2].…”

Section: Introductionmentioning

confidence: 99%