Structure Of Hemocyanin Research Articles

Ammonia stress affects the structure and function of hemocyanin in Penaeus vannamei

Anthropogenic factors and climate change have serious effects on the aquatic ecosystem and aquaculture. Among water pollutants, ammonia has the greatest impact on aquaculture organisms such as penaeid shrimp because it makes them more susceptible to infections. In this study, we explored the effects of ammonia stress (0, 50, 100, and 150 mg/L) on the molecular structure and functions of the multifunctional respiratory protein hemocyanin (HMC) in Penaeus vannamei. While the mRNA expression of Penaeus vannamei hemocyanin (PvHMC) was up-regulated after ammonia stress, both plasma hemocyanin protein and oxyhemocyanin (OxyHMC) levels decreased. Moreover, ammonia stress changed the molecular structure of hemocyanin, modulated the expression of protein phosphatase 2 A (PP2A) and casein kinase 2α (CK2α) to regulate the phosphorylation modification of hemocyanin, and enhanced its degradation into fragments by trypsin. Under moderate ammonia stress conditions, hemocyanin also undergoes glycosylation to improve its in vitro antibacterial activity and binding with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Staphylococcus aureus) bacteria, albeit differently. The current findings indicate that P. vannamei hemocyanin undergoes adaptive molecular modifications under ammonia stress enabling the shrimp to survive and counteract the consequences of the stress.

Participation of Carbohydrate Structures in the Formation of Complex Structure of Hemocyanins from Haliotis tuberculata

Glycosylation plays an important role in many important processes in organisms. Their participation in the construction of the tertiary structure and immunotherapeutic activity of the hemocyanin Rapana venosa (RvH) from the Black Sea has been presented. Based on these results, the importance of glycans in organization of the structure of hemocyanin from the abdominal mollusk Haliotis tuberculata (HtH) was also studied.
 After incubation of both isoforms HtH1 and HtH2 with yeast Saccharomyces cerevisiae and enzyme Zymolyase, fractions with lower molecular weight (MW) between 45 and 65 kDa, which corresponds to MW of functional units (FUs) of hemocyanins, was identified. A comparative analysis of 10% SDS PAGE assays confirmed the depolymerization of the subunits HtH1 and HtH2 due to the specific glycosidase in the yeast S. cerevisiae and the enzyme Zymolyase. The presented results confirm our hypothesis that glycans are involved in the formation of the structural subunits of HtH.

Hemocyanins of Muricidae: New \u2018Insights\u2019 Unravel an Additional Highly Hydrophilic 800\xa0kDa Mass Within the Molecule

Hemocyanins are giant oxygen transport proteins that freely float within the hemolymph of most molluscs. The basic quaternary structure of molluscan hemocyanins is a cylindrical decamer with a diameter of 35 nm which is built of 400 kDa subunits. Previously published results, however, showed that one out of two hemocyanin subunits of Rapana venosa encompasses two polypeptides, one 300 kDa and one 100 kDa polypeptide which aggregate to typical 4 MDa and 8 MDa hemocyanin (di-)decamer molecules. It was shown that the polypeptides are bound most probably by one or more cysteine disulfide bridges but it remained open if these polypeptides were coded by one or two genes. Our here presented results clearly showed that both polypeptides are coded by one gene only and that this phenomenon can also be found in the gastropod Nucella lapillus. Thus, it can be defined as clade-specific for Muricidae, a group of the very diverse Caenogastropoda. In addition, we discovered a further deviation of this hemocyanin subunit within both species, namely a region of 340 mainly hydrophilic amino acids (especially histidines and aspartic acids) which have not been identified in any other molluscan hemocyanin, yet. Our results indicate that, within the quaternary structure, these additional amino acids most probably protrude within the inner part of didecamer cylinders, forming a large extra mass of up to 800 kDa. They presumably influence the structure of the protein and may affect the functionality. Thus, these findings reveal further insights into the evolution and structures of gastropod hemocyanins.

De Novo Structural Determination of the Oligosaccharide Structure of Hemocyanins from Molluscs.

A number of studies have shown that glycosylation of proteins plays diverse functions in the lives of organisms, has crucial biological and physiological roles in pathogen–host interactions, and is involved in a large number of biological events in the immune system, and in virus and bacteria recognition. The large amount of scientific interest in glycoproteins of molluscan hemocyanins is due not only to their complex quaternary structures, but also to the great diversity of their oligosaccharide structures with a high carbohydrate content (2–9%). This great variety is due to their specific monosaccharide composition and different side chain composition. The determination of glycans and glycopeptides was performed with the most commonly used methods for the analysis of biomolecules, including peptides and proteins, including Matrix Assisted Laser Desorption/Ionisation–Time of Flight (MALDI-TOF-TOF), Liquid Chromatography - Electrospray Ionization-Mass Spectrometry (LC/ESI-MS), Liquid Chromatography (LC-Q-trap-MS/MS) or Nano- Electrospray Ionization-Mass Spectrometry (nano-ESI-MS) and others. The molluscan hemocyanins have complex carbohydrate structures with predominant N-linked glycans. Of interest are identified structures with methylated hexoses and xyloses arranged at different positions in the carbohydrate moieties of molluscan hemocyanins. Novel acidic glycan structures with specific glycosylation positions, e.g., hemocyanins that enable a deeper insight into the glycosylation process, were observed in Rapana venosa, Helix lucorum, and Haliotis tuberculata. Recent studies demonstrate that glycosylation plays a crucial physiological role in the immunostimulatory and therapeutic effect of glycoproteins. The remarkable diversity of hemocyanin glycan content is an important feature of their immune function and provides a new concept in the antibody–antigen interaction through clustered carbohydrate epitopes.

The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase

Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc from Panulirus japonicus (PjHc) at 1.58 Å resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a “blocker residue” near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins.

Superexchange mechanism and quantum many body excitations in the archetypal di-Cu oxo-bridge

The hemocyanin protein binds and transports molecular oxygen via two copper atoms at its core. The singlet state of the {{rm{Cu}}}_{2}{{rm{O}}}_{2} core is thought to be stabilised by a superexchange pathway, but detailed in situ computational analysis is complicated by the multi-reference character of the electronic ground state. Here, electronic correlation effects in the functional site of hemocyanin are investigated using a novel approach, treating the localised copper 3d electrons with cluster dynamical mean field theory. This enables us to account for dynamical and multi-reference quantum mechanics, capturing valence and spin fluctuations of the 3d electrons. Our approach explains the stabilisation of the experimentally observed di-Cu singlet for the butterflied {{rm{Cu}}}_{2}{{rm{O}}}_{2} core, with localised charge and incoherent scattering processes across the oxo-bridge that prevent long-lived charge excitations. This suggests that the magnetic structure of hemocyanin is largely influenced by the many-body corrections.

Spectroscopic Studies on the Conformational Stability of Hemocyanin of Pila virens (Lamarck, 1822) in the Presence of Temperature and Detergents

Gastropods are endowed with a soluble copper containing, blue coloured respiratory protein known as hemocyanin. In the present study, for the first time, hemocyanin from Pila virens, a fresh water gastropod endemic to Indian sub-continent was studied for its conformational stability in the presence of temperature and various detergents by different spectroscopic techniques viz. UV–Vis, fluorescence and circular dichroism spectroscopy. Results from UV–Vis spectroscopy revealed that, absorbance corresponding to active site moieties were decreased with increase in temperature from 30 to 70 °C. Whereas ‘thermal reversibility’ studies showed the increase in absorbance of active site with decrease in temperature from 70 to 20 °C (is an indicative of the characteristic feature of hemocyanins—that oxygen is reversibly bound to the copper in the active site). Results from CD spectroscopy revealed that the temperature-induced conformational changes on the secondary structure of hemocyanin was reversible up to 70 °C. Beyond 80 °C, the protein was completely denatured and assumed an increased random coil conformation. Hemocyanin in the presence of high concentration of detergents lost its structure as evident from UV–Vis fluorescence (evident from the red shifts in the emission maxima accompanied by quenching in emission intensities) and CD spectroscopy. Stability of active site moieties in the presence of different detergents was also studied by UV–Vis spectroscopy. The biophysical techniques gave insights into the varied effects of each of the different denaturants on the conformational stability of hemocyanin from Pila virens.

Jumping on the Edge-First Evidence for a 2 × 6-meric Hemocyanin in Springtails.

Hemocyanins are respiratory dioxygen carrier proteins found in many arthropods including ancient terrestrial species such as spiders and scorpions as well as marine horseshoe crabs. As hemocyanins are highly conserved in this lineage, it is possible to observe an evolutionary descent through its subunits and their overall structure. Unfortunately, little is known about the structure and function of hexapod hemocyanins. Using recent springtail taxa (Collembola) as models for basal hexapods, and the help of electron microscopy, light scattering, SDS PAGE, and Western blot, we could demonstrate for the first time the presence of 2 × 6-meric hemocyanins in the hemolymph of hexapods. The quaternary structure is composed of at least two different subunits and looks nearly identical to the hemocyanin found in decapod crustaceans. In addition, homology modeling and western blotting suggest a close structural relationship between collembolan and crustacean hemocyanin. Such a respiratory protein was possibly helpful in the early terrestrialization process of ancient Collembola. In addition, physiological adaptations to hypoxic or temporarily anoxic conditions could be a possible explanation for the presence of this respiratory protein. Nevertheless, it has to be concluded that the primary benefit of hemocyanin for springtails remains unclear.

Cryo-EM reveals the asymmetric assembly of squid hemocyanin.

The oxygen transporter of molluscs, hemocyanin, consists of long pearl-necklace-like subunits of several globular domains. The subunits assemble in a complex manner to form cylindrical decamers. Typically, the first six domains of each subunit assemble together to form the cylinder wall, while the C-terminal domains form a collar that fills or caps the cylinder. During evolution, various molluscs have been able to fine-tune their oxygen binding by deleting or adding C-terminal domains and adjusting their inner-collar architecture. However, squids have duplicated one of the wall domains of their subunits instead. Here, using cryo-EM and an optimized refinement protocol implemented in SPHIRE, this work tackled the symmetry-mismatched structure of squid hemocyanin, revealing the precise effect of this duplication on its quaternary structure and providing a potential model for its structural evolution.

Structure, diversity and evolution of myriapod hemocyanins

Oxygen transport in the hemolymph of many arthropods is mediated by hemocyanins, large copper-containing proteins that are well-studied in Chelicerata and Crustacea, but had long been considered unnecessary in the subphylum of Myriapoda. Only recently has it become evident that hemocyanins are present in Scutigeromorpha (Chilopoda) and Spirostreptida (Diplopoda). Here we present evidence for a more widespread occurrence of hemocyanin in the myriapods. By means of RT-PCR, western blotting and database searches, hemocyanins were identified in the symphylans Hanseniella audax and Symphylella vulgaris, the chilopod Scolopendra subspinipes dehaani and the diplopod Polydesmus angustus. No hemocyanins were found in the diplopods Polyxenus lagurus, Cylindroiulus punctatus, Glomeris marginata, Glomeris pustulata and Arthrosphaera brandtii, or the chilopods Lithobius forficatus, Geophilus flavus and Strigamia maritima. This suggests multiple independent losses in myriapod taxa. Two independent hemocyanin subunits were found that were already present in the myriapod stem line. We specifically investigated the structure of the hemocyanin of P. angustus, which consists of three distinct subunits that occur in an approximately equimolar ratio. As deduced by 3D electron microscopy, the quaternary structure is a 3 × 6-mer that resembles the half structure of the 6 × 6-mer hemocyanin from Scutigera coleoptrata. It was analyzed more closely by homology modeling of 1 × 6-mers and their rigid-body fitting to the electron density map of the 3 × 6-mer. In addition, we obtained the cDNA sequence of a putative myriapod phenoloxidase. Phenoloxidases are related to the arthropod hemocyanins, but diverged before radiation of the arthropod subphyla.

Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes

Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the β ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. β ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MDa.

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 35nm 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 550kDa. 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.

ESI-MS and MALLS analysis of quaternary structure of molluscan hemocyanins

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)) and multi-angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3 kDa, respectively) and S. officinalis (4134 and 443.8 kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0 kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK-trypsin. Eight functional units (FUs) with masses of ~ 50 kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI-mass spectrometry (MS) and MALDI-MS, methods gave insights into post-translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0 kDa, respectively) between the masses measured by ESI-MS and those calculated by their gene sequences.

Crystallization and Preliminary Analysis of Crystals of the 24-Meric Hemocyanin of the Emperor Scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (MW = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.

Homology models of four Agaricus bisporus tyrosinases

Partially purified tyrosinase from the white button mushroom Agaricus bisporus is available commercially and is a widely used experimental model for the study of tyrosinase. The structure of an H2L2 tetrameric form of the mushroom enzyme was recently determined by X-ray crystallography. In this structure the two H subunits originate from the PPO3 gene, and the two L subunits are formed by a protein of unknown function with a lectin-like fold. However, the X-ray structures and oligomeric states of the mushroom PPO1, PPO2, PPO4, and PPO5 gene products remain unknown. Commercial mushroom tyrosinase powder is a mixture containing several or all of these tyrosinases, so knowledge of their structures should provide insight regarding interpretation of experimental data generated using commercial preparations of the enzyme. The PPO3 structure (H-subunit) was used as a template to generate homology models for the structures of the other four tyrosinases, and the resulting structural models were evaluated. Due to the moderate to high percentage of sequence identity (∼37–76%) between PPO3 and the other four tyrosinases, the backbone conformations of the predicted structures are very similar to that of PPO3. The alpha carbons of the six copper-coordinating histidines in the active site are positioned properly in the predicted structures, but their side chains are not oriented optimally for copper binding in some cases. Thus, the models are likely to provide an accurate representation of the actual tertiary structures, but they may have limited use in studies involving docking of substrates or inhibitors in the active site. Comparison of the homology models to the structure of molluscan hemocyanin enabled a prediction of the orientation of the enzyme's C-terminal domain over the active site in the latent enzyme.

Structure of hemocyanin from garden snail Helix lucorum

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as beta-HlH, alpha(D)-HlH and alpha(N)-HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (beta-HlH) and 1079 kDa (alpha(D)-HlH, and alpha(N)-HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl(2). All isoforms were reassociated into didecamers and tubules with different length, but in contrast to alpha(D)-HlH isoform, longer tubules were observed in beta-HlH. Moreover the structure of beta-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes.

Environmental and Physiological Controls of Blue Crab Avoidance Behavior During Exposure to Hypoxia

Generalizing the impacts of hypoxia on aquatic animal populations is difficult due to differences in behavioral and physiological responses among individuals as well as varying hydrodynamics of hypoxic events. Information on which environmental cues animals use to avoid hypoxia and how abiotic covariates and physiology influence avoidance behavior is lacking. Our laboratory flume studies quantified the interacting effects of hydrography (dissolved oxygen [DO], temperature, and salinity), hydrodynamics (rate of DO decline and current speed), and physiological state on avoidance behaviors of blue crabs (Callinectes sapidus). Changes in DO stimulated increased rates of movement, regardless of whether the change resulted in hypoxia. Increased rates of DO decline stimulated faster movement rates under hypoxic conditions because crabs spent less time in hypoxia compared to crabs under conditions of slow rate of DO decline. Blue crabs that had hemocyanin structures with a high affinity for O(2) (hypoxia-tolerant) were less active under hypoxic conditions than conspecifics with hemocyanins with a low O(2) affinity, suggesting that physiological state influences behavioral responses to stressors. These results provide a mechanistic understanding of how physiological acclimation and hypoxia hydrodynamics may influence population dynamics.

Keyhole Limpet Hemocyanin: 9-Å CryoEM Structure and Molecular Model of the KLH1 Didecamer Reveal the Interfaces and Intricate Topology of the 160 Functional Units

Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-Å cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod hemocyanin decamer [ Nautilus pompilius hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five “arcs” (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod hemocyanin didecamer assembled from two asymmetric decamers [isoform keyhole limpet hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five “slabs” (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-Å data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod hemocyanin and essentially solve the gastropod hemocyanin structure.

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-Å 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.

Comparative 11 Å structure of two molluscan hemocyanins from 3D cryo-electron microscopy

Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350–400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O 2 molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we show 3D reconstructions from cryo-electron micrographs of the hemocyanin decamer of Nautilus pompilius (Cephalopoda) and Haliotis tuberculata (Gastropoda) at a resolution of 11 Å (FSC 1/2-bit criterion). The wall structure of both hemocyanins is very similar and shows, as in previous reconstructions, three tiers with 20 functional units each that encircle the cylinder wall, and the 10 oblique minor and major wall grooves. However, the six types of wall FUs of the polypeptide subunit, termed a-b-c-d-e-f, are now for the first time individually discernable by their specific orientation, shape, and connections. Also, the internal collar complex of the decamers shows superior resolution which, in this case, reveals striking differences between the two hemocyanins. The five arcs (FU-g pairs) of the central collar (in both hemocyanins) and the five slabs (FU-h pairs) of the peripheral collar (only present in Haliotis hemocyanin), as well as their connections to the wall and to each other are now more clearly defined. The arc is attached to the wall through a feature termed the anchor, a previously undescribed structural element of the hemocyanin wall.