Complete amino-acid sequence of a functional unit from a molluscan hemocyanin (Helix pomatia)

From the beta c-hemocyanin (beta c-Hc) of the vineyard snail, Helix pomatia, the functional unit d (Mr approximately equal to 50,000-55,000) was isolated by limited proteolysis and gel chromatography. A small quantity of functional unit d was obtained intact, but the major part in the form of two peptides (Mr approximately equal to 43,000 and 10,000, respectively) connected by a disulfide bridge. After reduction and carboxymethylation, these were separated from each other and cleaved by conventional methods. The peptides were isolated by gel chromatography and HPLC, and sequenced manually or automatically. The complete sequence of Helix beta c-Hc d comprises 410 residues plus 3 residues at the N-terminus seemingly resulting from incomplete cleavage. There is apparently only one carbohydrate side-chain. Comparison of this gastropodan hemocyanin sequence to the partial sequence of a cephalopodan Hc C-terminal unit revealed sufficient identities to state that the functional units of molluscan hemocyanins have arisen by a series of gene duplications. On the other hand, there is practically no homology with arthropodan hemocyanins except for one section of 42 residues which is clearly homologous. This section corresponds to the "Copper B" site of Panulirus interruptus hemocyanin. It is also found in tyrosinases from Neurospora crassa, Streptomyces glaucescens, and mouse. In the N-terminal half of Helix beta c-Hc d there are other sections clearly homologous to the tyrosinases, but overall homology is limited. The second copper-binding site was not identified but must be completely distinct from the "Copper A" binding site of arthropodan hemocyanins. It is suggested that molluscan and arthropodan hemocyanins have evolved independently from a common ancestral mononuclear copper protein.     

Molecular evolution of the arthropod hemocyanin superfamily

Arthropod hemocyanins are members of a protein superfamily that also comprises the arthropod phenoloxidases (tyrosinases), crustacean pseudohemocyanins (cryptocyanins), and insect storage hexamerins. The evolution of these proteins was inferred by neighbor-joining, maximum-parsimony, and maximum-likelihood methods. Monte Carlo shuffling approaches provided evidence against a discernible relationship of the arthropod hemocyanin superfamily and molluscan hemocyanins or nonarthropodan tyrosinases. Within the arthropod hemocyanin superfamily, the phenoloxidase probably emerged early in the (eu-)arthropod stemline and thus form the most likely outgroup. The respiratory hemocyanins evolved from these enzymes before the radiation of the extant euarthropodan subphyla. Due to different functional constraints, replacement rates greatly vary between the clades. Divergence times were thus estimated assuming local molecular clocks using several substitution models. The results were consistent and indicated the separation of the cheliceratan and crustacean hemocyanins close to 600 MYA. The different subunit types of the multihexameric cheliceratan hemocyanin have a rather conservative structure and diversified in the arachnidan stemline between 550 and 450 MYA. By contrast, the separation of the crustacean (malacostracan) hemocyanin subunits probably occurred only about 200 MYA. The nonrespiratory pseudohemocyanins evolved within the Decapoda about 215 MYA. The insect hemocyanins and storage hexamerins emerged independently from the crustacean hemocyanins. The time of divergence of the insect proteins from the malacostracan hemocyanins was estimated to be about 430-440 MYA, providing support for the notion that the Hexapoda evolved from the same crustacean lineage as the Malacostraca.     

Minireview: Recent progress in hemocyanin research

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-? structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.     

Hexamers of subunit II from Limulus hemocyanin (a 48-mer) have the same quaternary structure as whole Panulirus hemocyanin molecules

Hemocyanins are copper-containing proteins that transport oxygen in a variety of invertebrates. Considerable evidence has accumulated that arthropodan hemocyanins are multimers of a fundamental hexameric unit. X-Ray crystallographic structure determination has revealed that the hemocyanin molecule from the spiny lobster Panulirus interruptus is a single hexamer having 32 point group symmetry. Using crystals of subunit II, one of 8 polypeptide types comprising the octahexameric hemocyanin of the horseshoe crab Limulus polyphemus, and the molecular replacement method for crystallographic phase determination we show that subunit II forms assemblies with the same hexameric quaternary structure as the whole Panulirus hemocyanin molecule. Observation of the same hexameric motif in two widely separated species provides strong additional evidence that this quaternary structural unit is a universal building block of arthropodan hemocyanins.     

Common origin of arthropod tyrosinase,arthropod hemocyanin,insect hexamerin,and dipteran arylphorin receptor

Dipteran arylphorin receptors, insect hexamerins, cheliceratan and crustacean hemocyanins, and crustacean and insect tyrosinases display significant sequence similarities. We have undertaken a systematic comparison of primary and secondary structures of these proteins. On the basis of multiple sequence alignments the phylogeny of these proteins was investigated. Hexamerin subunits, hemocyanin subunits, and tyrosinases share extensive similarities throughout the entire amino acid sequence. Our studies suggest the origin of arthropod hemocyanins from ancient tyrosinase-like proteins. Insect hexamerins likely evolved from hemocyanins of ancient crustaceans, supporting the proposed sister-group position of these subphyla. Arylphorin receptors, responsible for incorporation of hexamerins into the larval fat body of diptera, are related to hexamerins, hemocyanins, and tyrosinase. The receptor sequences display extensive similarities to the first and third domains of hemocyanins and hexamerins. In the middle region only limited amino acid conservation was observed. Elements important for hexamer formation are deleted in the receptors. Phylogenetic analysis indicated that dipteran arylphorin receptors diverged from ancient hexamerins, probably early in insect evolution. Correspondence to: T. Burmester     

A Cladistic Analysis of the Evolutionary Relationships of the Members of the Tyrosinase Gene Family Using Sequence Data

Recently, DNA sequence data have been published on tyrosinase and tyrosinase-related proteins (TRPs) in a wide variety of vertebrates ranging from Rana to Homo. These proteins are in turn members of a larger family of binuclear copper-binding proteins, which all contain two highly conserved copper-binding domains. This gene family also includes tyrosinases from fungi and bacteria as well as arthropodan and molluscan hemocyanins. Parsimony-based alignment and tree construction algorithms (Malign, vl.85 and PAUP, 3.1.1) were used to analyze the diversification of both the evolutionarily conserved copper-binding domains i6n copper-binding proteins in general as well as the diversification of the vertebrate tyrosinase gene family more specifically. These analyses show that the diversification of the vertebrate tyrosinase gene family minimally predates the diversification of vertebrates. Vertebrate tyrosinases proper first diverged from an ancestral tyrosinase-related protein (TRP) that then subsequently diverged to form tyrosinase-related protein-Is (TRP-1s) and tyrosinase-related protein-2s (TRP-2s).     

Molluscan hemocyanin: structure,evolution, and physiology

Most molluscs have blue blood because their respiratory molecule is hemocyanin, a type-3 copper-binding protein that turns blue upon oxygen binding. Molluscan hemocyanins are huge cylindrical multimeric glycoproteins that are found freely dissolved in the hemolymph. With molecular masses ranging from 3.3 to 13.5 MDa, molluscan hemocyanins are among the largest known proteins. They form decamers or multi-decamers of 330- to 550-kDa subunits comprising more than seven paralogous functional units. Based on the organization of functional domains, they assemble to form decamers, di-decamers, and tri-decamers. Their structure has been investigated using a combination of single particle electron cryo-microsopy of the entire structure and high-resolution X-ray crystallography of the functional unit, although, the one exception is squid hemocyanin for which a crystal structure analysis of the entire molecule has been carried out. In this review, we explain the molecular characteristics of molluscan hemocyanin mainly from the structural viewpoint, in which the structure of the functional unit, architecture of the huge cylindrical multimer, relationship between the composition of the functional unit and entire tertiary structure, and possible functions of the carbohydrates are introduced. We also discuss the evolutionary implications and physiological significance of molluscan hemocyanin.     

Spider hemocyanin binds ecdysone and 20-OH-ecdysone

Fluorescence quenching studies and binding experiments with [(3)H]ecdysone reveal that the respiratory protein, hemocyanin, of the tarantula Eurypelma californicum binds ecdysone. The binding constant for ecdysone ranges between 0.5 and 5 mM, indicating a low affinity binding. However, it is comparable with those found for the ecdysone binding to hexamerins from insects. Based on a comparison of sequences and x-ray structures of arthropodan hemocyanins, we propose an evolutionary conserved hydrophobic pocket in domain 1 of the hemocyanin subunit that may bind ecdysone.     

Contribution of disulfide bonds and calcium to Molluscan hemocyanin stability

Disulfide bonds and calcium ions contribute significantly to the stability of the hemocyanin from the mollusc Rapana thomasiana grosse (gastropod). An extremely powerful protective effect of Ca2+ at a concentration of 100 mM (100% protection) against the destructive effect of reductants like dithiothreitol was observed. This is important for the practical application of molluscan hemocyanins in experimental biochemistry, immunology and medicine. The reduction of the disulfide bonds in the Rapana hemocyanin leads to a 20% decrease of the a-helical structure. The S-S bonds contribute significantly to the free energy of stabilization in water increasing delta G(D)H2O by 6.9 kJ mol (-1) The data are related to the X-ray model of the Rapana hemocyanin functional unit RtH2e. The results of this study can be of common validity for related respiratory proteins because the cysteine residues are conserved in all sequences of molluscan hemocyanins published so far.     

The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity

Structure-function relationships in a molluscan hemocyanin have been investigated by determining the crystal structure of the Rapana thomasiana (gastropod) hemocyanin functional unit RtH2e in deoxygenated form at 3.38 A resolution. This is the first X-ray structure of an unit from the wall of the molluscan hemocyanin cylinder. The crystal structure of RtH2e demonstrates molecular self-assembly of six identical molecules forming a regular hexameric cylinder. This suggests how the functional units are ordered in the wall of the native molluscan hemocyanins. The molecular arrangement is stabilized by specific protomer-to-protomer interactions, which are probably typical for the functional units building the wall of the cylinders. A molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins is proposed on the basis of the molecular interactions between the protomers. In particular, the deoxygenated RtH2e structure reveals a tunnel leading from two opposite sides of the molecule to the active site. The tunnel represents a possible entrance pathway for dioxygen molecules. No such tunnels have been observed in the crystal structure of the oxy-Odg, a functional unit from the Octopus dofleini (cephalopod) hemocyanin in oxygenated form.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

Comparison of the X-ray absorption properties of the binuclear active site of molluscan and arthropodan hemocyanins

The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge (XANES) and for the first shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla differences. The XANES spectra of oxy-hemocyanins of the different species are remarkably similar, consistent with a very strongly conserved co-ordination geometry of the copper active site. In contrast, small but significant differences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy-arthropodan hemocyanins (with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This difference is tentatively assigned to a more planar geometry of the copper-ligands system in the arthropodan rather than in the molluscan proteins.The first shell analysis of the EXAFS modulation is consistent with the presence of n=3Nepsilon(2) imidazole nitrogens at an average distance of 1.92 +/- 0.03 A from copper in all the deoxy-hemocyanins investigated.Binding of dioxygen results for all hemocyanins in the increase of the number of first shell back-scattering atoms to n=5 with average distances of 1.93 A. Alternatively, by separating the contribution of Nepsilon(2) imidazole nitrogens and of peroxide O-atoms, n=3 ligands at 1.98 +/- 0.03 A and n=2 ligands at 1.87 +/- 0.03 A are found.     

Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda

Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical confidence the integrity of the Tracheata (i.e., Myriapoda + Insecta) but give conflicting results on the position of the myriapod hemocyanin. While distance matrix and maximum-likelihood methods support a basal position of the Spirostreptus hemocyanin with respect to the other hemocyanins, parsimony analysis suggests a sister group relationship with the chelicerate hemocyanins. The latter topology is also supported by a unique shared deletion of an alpha-helix. A common ancestry of Myriapoda and Chelicerata should be seriously considered.     

Evolution of molluscan hemocyanin structures

Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed of many protein subunits that in turn encompass subsets of distinct functional units. The structure and evolution of molluscan hemocyanin have been studied for decades, but it required the recent progress in DNA sequencing, X-ray crystallography and 3D electron microscopy to produce a detailed view of their structure and evolution. The basic quaternary structure is a cylindrical decamer 35 nm in diameter, consisting of wall and collar (typically at one end of the cylinder). Depending on the animal species, decamers, didecamers and multidecamers occur in the hemolymph. Whereas the wall architecture of the decamer seems to be invariant, four different types of collar have been identified in different molluscan taxa. Correspondingly, there exist four subunit types that differ in their collar functional units and range from 350 to 550 kDa. Thus, molluscan hemocyanin subunits are among the largest polypeptides in nature. In this report, recent 3D reconstructions are used to explain and visualize the different functional units, subunits and quaternary structures of molluscan hemocyanins. Moreover, on the basis of DNA analyses and structural considerations, their possible evolution is traced. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

The sequence of a gastropod hemocyanin (HtH1 from Haliotis tuberculata)

The eight functional units (FUs), a-h, of the hemocyanin isoform HtH1 from Haliotis tuberculata (Prosobranchia, Archaeogastropoda) have been sequenced via cDNA, which provides the first complete primary structure of a gastropod hemocyanin subunit. With 3404 amino acids (392 kDa) it is the largest polypeptide sequence ever obtained for a respiratory protein. The cDNA comprises 10,758 base pairs and includes the coding regions for a short signal peptide, the eight different functional units, a 3'-untranslated region of 478 base pairs, and a poly(A) tail. The predicted protein contains 13 potential sites for N-linked carbohydrates (one for HtH1-a, none for HtH1-c, and two each for the other six functional units). Multiple sequence alignments show that the fragment HtH1-abcdefg is structurally equivalent to the seven-FU subunit from Octopus hemocyanin, which is fundamental to our understanding of the quaternary structures of both hemocyanins. Using the fossil record of the gastropod-cephalopod split to calibrate a molecular clock, the origin of the molluscan hemocyanin from a single-FU protein was calculated as 753 +/- 68 million years ago. This fits recent paleontological evidence for the existence of rather large mollusc-like species in the late Precambrian.     

Effects of aplysia hemocyanin on the conductance of oxidized cholesterol black lipid membranes

Treating oxidized cholesterol black lipid membranes with Aplysia hemocyanin induces the formation of channels with two conductivity states; at the fundamental level of conductance, the lifetime is several hours. Transitions from this state to a different conductivity state occur. Membranes with many of these channels have a voltage-dependent conductance and transitions between different conductivity values occurring in a few ms. Thus molluscan hemocyanins can be considered as a general class of pore-forming proteins.     

Phenoloxidase activity of intact and chemically modified functional unit RvH1: a from molluscan Rapana venosa hemocyanin

o-Diphenol oxidase activities (o-diPO) of chemically modified functional unit RvH1-a of molluscan hemocyanin Rapana venosa were studied using L-Dopa and dopamine as substrates. With L-Dopa as substrate the native FU RvH1-a did not show any o-diPO activity. Therefore the native FU RvH1-a was converted to enzymatic active form, after treatment with SDS, trypsin, urea and different values of pH when its o-diPO activity was studied. The highest artificial induction of o-diPO activity was observed after incubation of FU with 3.0mM SDS, and RvH1-a shows both, dopamine (K(M)=6.53mM, k(cat)/K(M)=1.29) and L-Dopa (K(M)=2.0mM, k(cat)/K(M)=2.1) activity due to a more open active site of the enzyme and better access of the substrates. It was determined that the K(M) value of SDS-activated RvH1-a against dopamine is higher compared to those of hemocyanins from Helix vulgaris, Helix pomatia and native tyrosinase from Ipomoea batatas but much lower than that from Illex argentinus (ST94) tyrosinase and arthropodan hemocyanin from Carcinus aestuarii. The Km value of SDS-activated RvH1-a against L-Dopa is higher than those of hemocyanins from H. vulgaris and Cancer magister, but lower than that of the tyrosinase from Streptomyces albus.     

Complete subunit sequences, structure and evolution of the 6 x 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata.

Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6 x 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5-55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 x 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.     

Evolution of the arthropod prophenoloxidase/hexamerin protein family

 Phylogenetic analysis of the prophenoloxidase/hexamerin family of arthropods revealed four well supported subfamilies: (1) the arylphorin subfamily, including arylphorins, storage proteins, and other proteins of uncertain function from insects; (2) the hemocyanins of branchiopod crustaceans, which are copper-binding proteins involved in oxygen transport; (3) the hemocyanins of chelicerates; and (4) the prophenoloxidases (proPO) of both insects and branchiopods, which are copper-binding molecules that play a role in sclerotization of cuticle and encapsulation of foreign particles. The phylogeny indicated that insect and branchiopod proPO constitute a monophyletic group but that branchiopod and chelicerate hemocyanins do not constitute a monophyletic group. Branchiopod hemocyanin and proPO diverged from each other prior to the divergence of insects from branchiopods and probably prior to the divergence of chelicerates from the insect-branchiopod lineage. Likewise, the insect arylphorin subfamily diverged from proPO prior to the divergence of insects from branchiopods and probably prior to the divergence of chelicerates; thus, the results did not support the hypothesis that insect arylphorins represent hemocyanins freed to assume a new function because the insect tracheal respiratory system removes the need for an oxygen-transport molecule. Nonetheless, reconstruction of ancestral sequences by the maximum parsimony method suggested that the ancestors of the arylphorin family were copper-binding. Regions corresponding to the copper-binding domains were found to have a faster rate of nonsynonymous evolution in arylphorin subfamily genes than in other hexamerin family genes; this presumably reflects a relaxation of purifying selection after the loss of copper-binding function. Received: 25 March 1998 / Revised: 3 July 1998     

Conformational states of the Rapana thomasiana hemocyanin and its substructures studied by dynamic light scattering and time-resolved fluorescence spectroscopy

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.