Copper site of molluscan oxyhemocyanins. Structural evidence from x-ray absorption spectroscopy

Co, Man Sung; Hodgson, Keith O.; Eccles, T. K.; Lontie, René

doi:10.1021/ja00394a066

Cited by 87 publications

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“…1 is reasonably well defined on the basis ofour chemical, spectral, and theoretical studies (11,12) of the series of derivatives shown in this figure, as well as resonance Raman studies, which indicated (19) the peroxide oxidation state of the bound dioxygen, and extended x-ray absorption fine structure (EX-AFS) analysis, which gave a Cu-Cu distance of 3.67 A (20) or 3.55 A (21). Cu(I) has a tendency toward lower coordination numbers, softer ligands, and a geometry based on ligand-ligand replusion (rather than unpaired d electron effects) relative to Cu(II).…”

mentioning

confidence: 61%

Preparation of a spectral probe derivative of the hemocyanin biopolymer: effects of allosteric interactions on the coupled binuclear copper active site.

Hwang¹,

Solomon²

1982

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

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A series ofderivatives for both the arthropod and mollusc hemocyanin biopolymers has been prepared; the derivatives contain a small fraction ofelectron paramagnetic resonancedetectable half-met [Cu(fl) Cu(I)] sites dispersed among the nondetectable oxy binuclear copper active sites. Upon deoxygenation, large changes in the electron paramagnetic resonance signal of these half-met spectral probe derivatives are observed, which are further adjusted by the heterotropic effectors Ca2+ and HW. The active site structural changes indicated by these spectral changes as the hemocyanins go from a relaxed to a tensed quaternary structure are then discussed.Hemocyanins, the oxygen transport proteins in the hemolymph of molluscs and arthropods, reversibly bind dioxygen with a stoichiometry ofone dioxygen per two copper ions. Under physiological conditions, the hemocyanins are present as highly aggregated biopolymers with a molecular architecture that differs dramatically between the two phyla. For the arthropods, a single subunit has a molecular weight of "'70,000 and contains one binuclear copper active site. At pH values below 8 and in the presence of Ca2+, these subunits aggregate into one, two, four, and in the case of Limulus polyphemus, up to eight hexamers.Alternatively, the smallest subunit in the molluscs contains about eight binuclear copper active sites (domains) covalently linked in a single polypeptide chain of "'400,000 daltons. In the mollusc Busycon canaliculatum, at pH less than 8, twenty chains aggregate to form a whole molecule that contains 160 active sites and appears as a cylindrical barrel under the electron microscope. When aggregated, the hemocyanins are highly cooperative in their oxygen binding, with Hill coefficients dependent on species, pH, and the presence of divalent cations (1,2).In general, current thermodynamic and kinetic theories of oxygen binding to the hemocyanins parallel [with small modifications (3, 4)] the allosteric model of hemoglobin. Deoxyhemocyanin has a "tensed" quaternary structure with a low oxygen affinity. Initial binding of oxygen to a limited number of active sites alters the successive oxygen affinities ofthe remaining sites by a ligand-induced conformational change of the protein (5) (i.e., oxygen is a homotropic effector); this results in the oxyhemocyanin having a "relaxed" quaternary structure with a high oxygen affinity. Protons and divalent cations act as heterotropic effectors, which, depending upon the species, stabilize either the tensed or relaxed quaternary structure.For hemoglobin, through both protein crystallography (6) and the detailed study of iron prophyrin model complexes (7), significant advances have been made in the development of a structural model that accounts for the active site changes related to these changes in oxygen affinity. Two basic spectroscopic approaches have been taken to relate this mechanical model to the hemoglobin tetramer. One approach involves the preparation of valence hybrids in which either the a or the (3 chains con...

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confidence: 61%

Preparation of a spectral probe derivative of the hemocyanin biopolymer: effects of allosteric interactions on the coupled binuclear copper active site.

Hwang¹,

Solomon²

1982

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

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“…The presence of sulfhydryl groups in the coordination sphere of the hemocyanin coppers (type 111) has been suggested by Klotz & Klotz (1955) and Amundsen et al (1977). In addition it has been shown that removal of oxygen from the hemocyanin oxygen binding site results in a considerable increase of the copper-copper distance concomitant with the breakage of a protein ligand bridge between the two coppers (Co & Hodgson, 1981;Brown et al, 1980). These data suggest that a cysteine sulfur forms a bridge between the two coppers in 50% of the oxygenated active sites of Callinecetes hemocyanin.…”

Section: B R O U W E R B O N a V E N T U R A A N D B O N A V E N mentioning

confidence: 90%

“…They are known to resemble tyrosinases (Jolley et al, 1974;Eickman et al, 1978), and their carbon monoxide and peroxide adducts may be analogous to some of the intermediate states of the copper containing subunits of cytochrome c oxidase. The oxygen binding sites of both mollusc and arthropod hemocyanins are similar to the extent that a binuclear copper center is directly liganded to amino acid side chains of the protein (Brown et al, 1980;Co & Hodgson, 1981; Co et ai., 1981;Eickman et al, 1979). The amino acids involved in the binding of the copper atoms to the protein are still incompletely known.…”

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confidence: 98%

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Heavy metal ion interactions with Callinectes sapidus hemocyanin: structural and functional changes induced by a variety of heavy metal ions

Brouwer¹,

Bonaventura²,

Bonaventura³

1982

Biochemistry

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Hemocyanins are oligomeric proteins that reversibly bind oxygen. The oxygen binding site is a binuclear copper center bound to the protein by amino acid side chains. The hemocyanin of the blue crab, Callinectes sapidus, occurs in vivo as a mixture of 25S dodecamers and 16S hexamers, whose oxygen binding properties are identical. Four heavy metals have been used as probes of structure and function in this hemocyanin system. Divalent cations of cadmium, copper, mercury, and zinc induced an indefinite self-association of the hemocyanin molecule. These higher ordered association states can be dissociated by ethylenediaminetetraacetic acid. Callinectes oxyhemocyanin possesses at least three mercury binding sites: (1) a sulfhydryl group which forms a mercaptide bond with a single mercuric ion, (2) a tryptophanyl side chain which forms a noncovalent 1:1 complex with mercuric ions with an association constant of 5.7 X 10(15) M-1, and (3) lower affinity site(s) involved in the self-association process also observed with cadmium, copper, and zinc. Sites 1 and 2 are most likely also involved in the binding of cadmium. Upon removal of oxygen from the active site of hemocyanin, an additional binding site becomes available for the reaction with mercury. Binding of mercury to this site leads to loss of one of the coppers from the binuclear oxygen binding site. Both the binuclear copper center and allosteric sites on the hemocyanin are affected by heavy metal binding. Cadmium and zinc ions increase the oxygen affinity; mercury and copper ions have the opposite effect. All four heavy metal ions decrease the degree of cooperative oxygen binding. The mercury-induced changes in oxygen binding by 25S Callinectes hemocyanin appear to be the result of that metal's interaction with the high-affinity tryptophan binding site. Mercury binding to the available sulfhydryl group in oxyhemocyanin occurs without functional consequences. Heavy metal, hydrogen, and chloride ions affect the affinity of the first or last oxygen molecules bound to the hemocyanin, which results in the appearance of multiple T (low oxygen affinity) and R (high oxygen affinity) states. Additionally, these ions shift the equilibrium between the low and high oxygen affinity states. The appearance of additional R states at high pH is accompanied by the cleavage of a tyrosine hydrogen bond.

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“…1) (10,(12)(13)(14). involving two imidazoles per copper, a peroxide bridge and a 1" general, systems of this sort have low room temperature protein ligand bridge in an approximately square planar strucmagnetic moments$ signalling exchange-coupled copper tenture (6). Further studies by Solomon et al suggest two copper tres and in cases where exchange integrals were measured the centres bridged by peroxide and some endogenous ligand with exchange was ~b~e r v e d to increase with increasing oxygen two imidazoles and a water molecule at each five-coordinate bridge copper centre in various mollusc and arthropod oxyhemoIn this report we describe the copper coordination chemistry cyanins.…”

Section: Introductionmentioning

confidence: 99%

Mononuclear and binuclear copper(II) complexes of some polyfunctional pyridyl phthalazines

Bullock¹,

Hartstock²,

Thompson³

1983

Can. J. Chem.

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. J. Chem. 61, 57 (1983).Mononuclear and binuclear copper(I1) complexes of the potentially tetradentatc binucleating ligands 1,4-di(3'-methyl-2'-pyridy1)aminophthalazine (PAP3Me) and 1,4-di(5'-methyl-2'-pyridyl)aminophthalazine (PAP5Me) are reported. In the case of the ligand 1,4-di(4',6'-dimethyl-2'-pyridyl)aminophthalazine (PAP46DiMe) only binuclear derivatives were obtained. ' The mononuclear complexes, involving protonated ligand, result from the sensitivity of these ligands to mildly acidic conditions in aqueous solutions of certain copper salts. Binuclear, neutral ligand, complexes are also formed when the pH increases. Hydroxy bridged binuclear structures exist in all the binuclear complexes described with the exception of one acetate bridged derivative. Sub normal magnetic moments are observed for all the binuclear complexes (< 1.74 BM) indicating spin coupled systems with very low values (<0.88 BM) for binuclear complexes of PAP46DiMe, indicating the possibility of strong antiferromagnetic exchange between the metal centres.

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Copper site of molluscan oxyhemocyanins. Structural evidence from x-ray absorption spectroscopy

Cited by 87 publications

References 0 publications

Preparation of a spectral probe derivative of the hemocyanin biopolymer: effects of allosteric interactions on the coupled binuclear copper active site.

Preparation of a spectral probe derivative of the hemocyanin biopolymer: effects of allosteric interactions on the coupled binuclear copper active site.

Heavy metal ion interactions with Callinectes sapidus hemocyanin: structural and functional changes induced by a variety of heavy metal ions

Mononuclear and binuclear copper(II) complexes of some polyfunctional pyridyl phthalazines

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