Oxygen Transport Protein Research Articles

The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins.

The crystal structure of catechol oxidase reveals new insight into the functional properties of the type-3 copper proteins. This class of proteins includes the closely related and better-known tyrosinase as well as hemocyanin, an oxygen transport protein. All these proteins have a dinuclear copper center, have similar spectroscopic behaviors, and show close evolutionary and functional relationships. Comparison between the 3D structures of catechol oxidase and hemocyanins reveals the structural reasons for the divergence in function.

Ontogeny of Crustacean Respiratory Proteins

The respiratory proteins hemoglobin and hemocyanin share the function of oxygen transport, but the proteins belong to separate gene families, and their active sites and the metal ions that bind the oxygen differ. Either hemoglobin or hemocyanin, but not both, is expressed in the hemolymph of many arthropod crustaceans. Hemoglobin is present in Branchiopoda, Ostracoda, Copepoda, rhizocephalan Cirripedia and one suborder of amphipodan Malacostraca, while hemocyanin has been described in Malacostraca. Recent work by several laboratories have provided new information on the gene structure, exon-intron patterns, site of synthesis and expression of hemoglobins in the branchiopods Artemia and Daphnia. These studies suggest the branchiopods are excellent model organisms for studies of oxygen sensors and hypoxia inducible transcription factors during developmental and adult stages. The focus in our laboratory on the ontogeny of hemocyanin in the Dungeness crab, Cancer magister, has demonstrated that both structure and function of hemocyanin change from megalopa to adult crab. The hemocyanin of an oceanic megalopa contains four subunits. Another subunit appears about the time of metamorphosis to first juvenile instar, and expression of a sixth subunit begins four or five molts later. The timing of onset of adult hemocyanin can be altered experimentally by food levels and temperature. Gene expression and functional properties of both red and blue oxygen transport proteins of crustaceans change during ontogeny to insure oxygen delivery appropriate for each developmental stage.

Aggregation of a crown ether-based copper amphiphile as a mimic for the superstructure of hemocyanin

Inspired by the alkali metal ion-controlled self-assembly of the oxygen-transporting dinuclear copper protein hemocyanin, a novel crown ether amphiphile (1) has been designed. A diaza-18-crown-6 moiety (diaza-18-crown-6 = 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane) is appended with an aliphatic C16 chain and with a PY2 ligand (PY2 = bis[2-(2-pyridyl)ethyl]amine), resulting in an amphiphile that has independent ligand sets for the coordination of copper ions and alkali metal ions. The copper complex of this amphiphile, [CuII(1)](ClO4)2, forms monolayers at the air–water surface. In the presence of certain alkali metal salts, the molecules in the monolayers stretch out and form sandwich complexes (2 ∶ 1 amphiphile : metal ion stoichiometry). This is in contrast to the 1 ∶ 1 complexes which are obtained with amphiphiles that lack the Cu–PY2 part. Electron microscopy experiments reveal that hollow tubules are generated when [CuII(1)](ClO4)2 is dispersed in water. The diameters of these tubes range from 45–55 nm, while their length can extend up to 5 μm. The presence of an alkali metal salt leads to vesicular structures with diameters ranging from 180–210 nm. Based on powder X-ray diffraction results, the amphiphile [CuII(1)](ClO4)2 is packed in strongly intercalated bilayers in both tubes and vesicles with a layer thickness of 4.8 nm. Cyclic voltammetry shows that the midpoint potential of aqueous dispersions of [CuII(1)](ClO4)2, E1/2 = −0.08 V, undergoes an anodic shift to +0.083 V in the presence of K+ ions. An in situ preparation of [CuI(1)](ClO4) did not show binding of molecular oxygen, whereas [CuII(1)](ClO4)2 reacted with H2O2, leading to degradation of ligand 1.

Quaternary Structure of Rice Nonsymbiotic Hemoglobin

Plant nonsymbiotic hemoglobins are hexacoordinate heme proteins found in all plants. Although expression is linked with hypoxic environmental conditions (Taylor, E. R., Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-862), no discrete physiological function has yet been attributed to this family of proteins. The crystal structure of a nonsymbiotic hemoglobin from rice has recently been determined. The crystalline protein is homodimeric and hexacoordinate with two histidine side chains coordinating the heme iron atom. Despite the fact that the amino acids responsible for the subunit interface are relatively conserved among the nonsymbiotic hemoglobins, previous work suggests that this group of proteins might display variability in quaternary structure (Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high pressure liquid chromatography were used to investigate the quaternary structure of rice nonsymbiotic hemoglobin at various states of ligation and oxidation. Additionally, site-directed mutagenesis was used to test the role of several interface amino acids in dimer formation and ligand binding. Results were analyzed in light of possible physiological functions and indicate that the plant nonsymbiotic hemoglobins are not oxygen transport proteins but more closely resemble known oxygen sensors.

Oxygen transport proteins: III. Structural studies of the scorpion ( Buthus sindicus) hemocyanin, partial primary structure of its subunit Bsin1

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the ‘top’ and the ‘side’ views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 ( M r=72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% α-helix, 18% β-sheet, 19% β-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed.

A Hemoglobin with an Optical Function

Hemoglobins are best known as oxygen transport proteins. Here we describe a hemoglobin from the parasitic nematode Mermis nigrescens (Mn-GLB-E) that has an optical, light shadowing function. The protein accumulates to high concentration as intracellular crystals in the ocellus of mature phototactic adult females while also being expressed at low concentration in other tissues. It differs in sequence and expression pattern from Mn-GLB-B, a second Mermis globin. It retains the structure and oxygen-binding and light-absorbing properties typical of nematode hemoglobins. As such, recruitment to a shadowing role in the eye appears to have occurred by changes in expression without modification of biochemistry. Both globins are coded by genes interrupted by two introns at the conserved positions B12.2 and G7.0, which is in agreement with the 3exon/2intron pattern model of globin gene evolution.

Disulfide Bond Reduction: A Powerful, Chemical Probe for the Study of Structure–Function Relationships in the Hemocyanins

The copper-containing hemocyanins are a class of oxygen-transport proteins whose structures differ in arthropods and molluscs. Crystal structure analyses and amino acid sequence comparisons show that disulfide bonding is a common feature in both arthropod and mollusc hemocyanins. Reduction of the disulfide bonds of a representative set of arthropod and mollusc hemocyanins results in complete loss of their oxygen-binding capacities. Thus, retention of the disulfide bonds is essential to the functional integrity of the oxygen-binding sites in the subunits of this class of oxygen carriers, despite the very different architectures of the arthropod and mollusc molecules. Depending upon the specific hemocyanin, partial to virtually complete restoration of the oxygen-binding capacity occurs when the disulfide-bond reductant is removed by dialysis. The rate at which the functional, active-site geometry is lost and the extent to which it can be restored varies markedly with hemocyanin type, aggregation state, and experimental conditions. Consequently, a comparison of these differences provides a simple, but powerful, way to probe internal and environmental factors that govern physiologically important structure–function relationships in this entire class of oxygen-transport proteins.

Dynamics of carbon monoxide binding with neuronal nitric oxide synthase.

The dynamics of CO rebinding with neuronal NO synthase (nNOS) following laser flash photolysis have been investigated from 293 to 77 K in the absence and presence of its substrate L-arginine. The distribution functions of the rate parameters P(k) and of the activation enthalpy P(H) were determined using the maximum entropy method. In a fluid solvent near room temperature, bimolecular rebinding is biphasic, as previously reported by several groups. However, measurement of the rotational correlation time shows that the apparent biphasic rebinding is not relevant to the genuine dynamics of NOS. In addition to native dimeric nNOS, another species (possibly aggregated or partially unfolded conformation) with different hydrodynamic characteristics is responsible for the faster rebinding process. In a rigid environment at low temperature, the geminate internal rebinding is not affected by the presence of the nonnative species. nNOS exhibits a bimodal distribution of CO activation enthalpy with P(H) consisting of two distinct bands with temperature-dependent amplitudes down to 77 K. The similarity of these findings with those recently reported for cytochromes P-450 suggests a common hierarchical organization of conformational substates, with a splitting of each conformational substate into a doublet. Thus, thiolate-coordinated heme proteins are in clear contrast to histidine-coordinated oxygen-transport heme proteins. The present results with nNOS provide additional support to previous arguments incriminating the thiolate ligand as responsible for the splitting of conformational substates.

Cryptocyanin, a crustacean molting protein: evolutionary link with arthropod hemocyanins and insect hexamerins.

Cryptocyanin, a copper-free hexameric protein in crab (Cancer magister) hemolymph, has been characterized and the amino acid sequence has been deduced from its cDNA. It is markedly similar in sequence, size, and structure to hemocyanin, the copper-containing oxygen-transport protein found in many arthropods. Cryptocyanin does not bind oxygen, however, and lacks three of the six highly conserved copper-binding histidine residues of hemocyanin. Cryptocyanin has no phenoloxidase activity, although a phenoloxidase is present in the hemolymph. The concentration of cryptocyanin in the hemolymph is closely coordinated with the molt cycle and reaches levels higher than hemocyanin during premolt. Cryptocyanin resembles insect hexamerins in the lack of copper, molt cycle patterns of biosynthesis, and potential contributions to the new exoskeleton. Phylogenetic analysis of sequence similarities between cryptocyanin and other members of the hemocyanin gene family shows that cryptocyanin is closely associated with crustacean hemocyanins and suggests that cryptocyanin arose as a result of a hemocyanin gene duplication. The presence of both hemocyanin and cryptocyanin in one animal provides an example of how insect hexamerins might have evolved from hemocyanin. Our results suggest that multiple members of the hemocyanin gene family-hemocyanin, cryptocyanin, phenoloxidase, and hexamerins-may participate in two vital functions of molting animals, oxygen binding and molting. Cryptocyanin may provide important molecular data to further investigate evolutionary relationships among all molting animals.

Crystal structure of a functional unit from Octopus hemocyanin

Hemocyanins are giant oxygen transport proteins found in many arthropods and molluscs. Freely dissolved in the hemolymph, they are multi-subunit proteins that contain many copies of the active site, a copper atom pair that reversibly binds oxygen. Octopus hemocyanin is composed of ten subunits, each of which contain seven oxygen-binding “functional units”. The carboxyl-terminal 47 kDa functional unit, Odg, is a proteolytic isolate that binds oxygen reversibly while exhibiting slight Bohr and magnesium ion effects. In this work we present the X-ray structure determination and analysis of Odg at 2.3 Å resolution. Odg has two structural domains: a largely α-helical copper binding domain, and a five-stranded anti-parallel β-sandwich with the jelly roll topology found in many viruses. Six histidine residues ligate the copper atoms, one of which is involved in a thioether bridge. The results show that the hemocyanin from the mollusc and that from the arthropod have distinct tertiary folds in addition to the long recognized differences in their quaternary structures. Nonetheless, a comparison of Octopus and horseshoe crab hemocyanin reveals a similar active site, in a striking example of perhaps both convergent and divergent evolution.

Molecular characterization and phylogenetic relationships of a protein with potential oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects?

Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.

Functional adaptations of oxygen-transport proteins.

Oxygen-transport proteins are multisubunit, circulating molecules that provide an efficient supply of oxygen to metabolically active metazoans. Hemoglobins, hemerythrins and hemocyanins have evolved in both structural and functional diversity and exhibit functional repertoires beyond that of simple, monomeric tissue myoglobins. Their phylogenetic distribution is intriguing, especially with respect to those organisms that express more than one type of oxygen-transport protein. An animal can modify the delivery of oxygen to its tissues by varying the rate of synthesis of these proteins or by selective expression of individual subunits and/or molecules. Changes in levels of allosteric modifiers that affect the protein's oxygenation properties will also modify oxygen delivery; some organisms have more ability than others to control concentrations of modulators. Hemoglobins have assumed functions in addition to oxygen transport, while hemocyanins have diversified through multiple gene duplications and functional specializations. Understanding the mechanisms of regulation of expression, synthesis and modulator levels is a key focus of current investigations.

Rapana thomasiana grosse (gastropoda) haemocyanin: spectroscopic studies of the structure in solution and the conformational stability of the native protein and its structural subunits

1. The stability towards pH changes, thermal and chemical (guanidine hydrochloride) denaturation of the oxy- and apo-forms of the native Rapana thomasiana haemocyanin and its structural subunits, RHSS1 and RHSS2, has been investigated using fluorescence and CD spectroscopy. The association of the subunits into haemocyanin aggregates increases considerably the melting temperature and the free energy of stabilization in water. The guanidine hydrochloride denaturation of the aggregated oxygen-transporting protein depends slightly on the protein concentration. The denaturation of the individual subunits is concentration-independent. Rapana haemocyanin is 5.9-7.5 kJ/mol more stable than the constituent polypeptide chains. 2. Upon excitation of the native haemocyanin and the subunits at 295 or 280 nm the fluorescence emission is determined by tryptophyl residues 'buried' deeply in the hydrophobic interior of the protein globules. This is confirmed by quenching experiments with acrylamide, caesium and iodide ions. The efficiency of the radiationless energy transfer between the phenol (donor) and indole (acceptor) fluorophores in the three species, native haemocyanin, RHSS1 and RHSS2, has been determined. An efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs in the non-dissociated form of the haemocyanin. 3. The tryptophan emission of the oxyhaemocyanin, oxy-RHSS1 and oxy-RHSS 2 is strongly quenched by the copper-dioxygen complex at the active site and the respective quantum yields of fluorescence of the oxygenated species are 4-7 times lower than those of the apo-forms. Protonated imidazole groups quench the fluorescence of neighbouring exited indole rings, probably by charge-transfer complex formation.

Identification of the Major Lipoproteins in Crayfish Hemolymph as Proteins Involved in Immune Recognition and Clotting

Lipid-containing hemolymph proteins from males of the crayfish Pacifastacus leniusculus were isolated by density gradient ultracentrifugation. Two major lipoproteins, one high density lipoprotein (HDL) and one very high density lipoprotein (VHDL), were characterized. The HDL and the VHDL were found to be identical to two proteins previously studied for their roles in immune recognition and hemolymph clotting, namely the β-1,3-glucan binding protein and the clotting protein. These results imply that crayfish lipoproteins have dual functions, and that they are involved in immunity, hemolymph clotting, and lipid transport in these animals. Also, the oxygen-transporting protein hemocyanin was found to have a small lipid content.

Oxygen transport proteins: I. Structure and organization of hemocyanin from scorpion (Buthus sindicus)

Scorpions are regarded as the oldest terrestrial arthropods. Scorpion Buthus sindicus (Buthidae) is commonly found in Pakistan and the Mediterranean region. The hemolymph of most arthropods contains large multisubunit, extracellular metalloprotein commonly known as hemocyanin which performs the important function of oxygen transport. The literature available to date shows that no attempt has been made to study hemocyanins or hemolymph proteins from this species. This communication presents the isolation, characterization and partial structural studies on hemocyanin from scorpion Buthus sindicus. (1) The hemolymph was collected by cardiac puncture, centrifuged and subjected to polyacrylamide gel electrophoresis and isoelectric focussing. (2) Crude hemolymph was subjected to gel filtration and high performance ion-exchange chromatography. (3) Purified hemocyanin subunits Bsin 1, 2 and 3 have been analysed for their amino acid composition and N-terminal sequence. The sequence homology was determined by comparison with other arthropod hemocyanin. The results are discussed.

Random Library Of Myoglobin Mutants Probes Protein Properties

Chemists at Stanford University have created a random library of single amino acid mutants of myoglobin to discover new ligand binding pathways in the protein. In addition to providing new insights into the function of myoglobin—the oxygen storage and transport protein in muscle—the approach is likely to be useful in probing structure-function relationships in other proteins. Conceptually, this is a simple idea which had almost no chance of working, but it did, says Steven G. Boxer, the chemistry professor at Stanford who directed the research. The approach developed by Boxer and Xiaohua Huang, a graduate student, differs sharply from site-directed mutagenesis, the technique that has revolutionized the study of structure-function relationships in proteins over the past decade. In site-directed mutagenesis, the researcher selects a particular amino acid residue in a protein and systematically inserts different amino acids at that position. Characterizing the properties of the mutant proteins sheds light ...

Recent Structural Work on the Oxygen Transport Protein Hemocyanin

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRecent Structural Work on the Oxygen Transport Protein HemocyaninKaren A. Magnus, Hoa Ton-That, and Joan E. CarpenterCite this: Chem. Rev. 1994, 94, 3, 727–735Publication Date (Print):May 1, 1994Publication History Published online1 May 2002Published inissue 1 May 1994https://doi.org/10.1021/cr00027a009RIGHTS & PERMISSIONSArticle Views1565Altmetric-Citations317LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (5 MB) Get e-Alerts Get e-Alerts

Evolution of arthropod hemocyanins and insect storage proteins (hexamerins).

Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper-binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine-rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.

Intermediate states of assembly in the dissociation of gastropod hemocyanin by hydrostatic pressure.

Under physiological conditions, the oxygen-transport protein from the gastropod Megalobulimulus ovatus, an extracellular hemocyanin, is composed of 20 identical subunits organized into a cylindrical structure (M(r) 9 x 10(6); 100 S). It dissociates in the pressure range of 0.4-2.5 kbar, as observed by spectroscopic methods (light scattering and intrinsic fluorescence) and gel filtration. In contrast to what is seen with smaller proteins, especially dimers, the pressure-dissociation curves for hemocyanin show little dependence on concentration, suggesting that native hemocyanin exists as a population of molecules with different free energies of association. The pressure-induced dissociation results from an equilibrium in which each aggregate responds to pressure independently of the others and, at any given pressure, is in one of two states, whole or dissociated, which persists for long times when compared with the duration of the experiment. The subunit-subunit affinity of dissociated hemocyanin is much lower than that of associated subunits, suggesting that a conformational drift of monomers occurs. When hemocyanin undergoes dissociation in the absence of calcium and at high pH (> 7.2), a large fraction of the dissociated products changes to a conformation that generates stable intermediate states of assembly, lacking the ability to fully reassemble into decamers and didecamers. These intermediates consist primarily of dimers (M(r) 900,000), and they bind oxygen reversibly with a higher affinity than the native hemocyanin. The binding of calcium or protons changes the conformation back to the "associable" state, which finally generates the assembled structure. The dissociation process is highly reversible at low pH (6.8-6.0) or in the presence of millimolar concentrations of calcium. At pH 5.7, dissociation is negligible at pressures up to 2.5 kbar. A decrease in pH from 7.6 to 6.6 increases the half-dissociation pressure (p 1/2) by 1.3 kbar, corresponding to a stabilization of 1.35 kcal per mole of subunit. The effects of Ca2+ and H+ may mean that, in vivo, special ionic conditions or other factors are required to be present at the assembly sites of oligomeric proteins such as hemocyanin.

Design and synthesis of model systems for oxygen binding and activation in dinuclear copper proteins.

Following the spectroscopic1,2 and crystallographie3,6 characterization of the dinuclear copper site of hemocyanin, the coordination chemistry of models for such sites was developed7,8. Nature employs the dinuclear copper site for binding of dioxygen, as found in the hemocyanins, the oxygen transport proteins of molluscs and arthropods. It is also employed in enzymes, such as tyrosinase9, and, in combination with copper ions representing other types of biological copper, lacease10 and ascorbate oxidase11, for the activation of dioxygen. Interestingly, protons and chloride ions affect the cooperativity of the oxygen binding by Panulirus interruptus (spiny lobster)12 and Limulus polyphemus (horseshoe crab)13 hemocyanin, respectively, whereas the aggregation of Octopus dofleini (Pacific octopus) hemocyanin subunits to form a functional unit is controlled by magnesium ions14. Recent crystal structures and spectroscopic studies5,6,15 of oxy- and deoxy-hemocyanin under a variety of circumstances have given indications that these effects may operate at the level of control of the Cu-Cu distance.