Integrative and Comparative Biology Advance Access originally published online on July 27, 2007
Integrative and Comparative Biology 2007 47(4):631-644; doi:10.1093/icb/icm063
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Minireview: Recent progress in hemocyanin research
*Institute for Molecular Biophysics, Johannes Gutenberg University, 55099 Mainz, Germany; Institute of Zoology, Johannes Gutenberg University, 55099 Mainz, Germany
Correspondence: 1Email: hdecker{at}uni-mainz.de; markl{at}uni-mainz.de
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Synopsis |
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Synopsis
Cooperative and allosteric...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 x 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.
Hemocyanins are respiratory proteins occurring freely dissolved in the hemolymph of many arthropods and molluscs. Arthropod hemocyanins are found as single hexamers (1 x 6mers) or multiples of hexamers (2 x 6mers, 4 x 6mers, 6 x 6mers, 8 x 6mers). Each arthropod hemocyanin subunit (ca. 72 kDa) folds into three domains characterized by different folding motifs (Volbeda and Hol 1989
; Hazes et al. 1993; Magnus et al. 1994): domain I with five or six 
-helices; domain II with a four -helix bundle and the active site containing two copper ions; and domain III with a seven-stranded antiparallel ß-barrel. In contrast, molluscan hemocyanins are cylindrical decamers, didecamers or multidecamers of a ca. 350 or 400 kDa polypeptide subunit. This folds into a chain of seven or eight functional units (FUs). Each FU is composed of two different structural domains termed (from -helix domain) and ß (from ß-sandwich domain); domain folds into a four -helix bundle carrying the copper active site, and domain ß into a six-stranded anti-parallel ß-barrel (Cuff et al. 1998). The -domain of a molluscan hemocyanin FU corresponds functionally to domain II of an arthropod hemocyanin and the ß-domain to domain III, respectively. Figure 1 shows the different structural levels of both hemocyanin types.
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The oxygen-binding behavior of hemocyanins is usually characterized by a low to moderate oxygen affinity that can be modulated by a variety of factors, as an adaptation to the species-specific ecophysiology. Cooperativity of oxygen binding is comparatively low in molluscan hemocyanins (with Hill coefficients around 2), but can be exceptionally high in arthropod hemocyanins (with Hill coefficients up to 9) (Loewe 1978
; Decker and Sterner 1990; van Holde and Miller 1995).
Various review articles on the structure, evolution and function of hemocyanins are available (Ellerton and Ellerton 1982; van Holde and Miller 1982, 1995; Markl 1986; Salvato and Beltramini 1990; Markl and Decker 1992; Solomon et al. 1994; Harris and Markl 1999; van Holde et al. 2001; Burmester 2001, 2002; Decker and Jaenicke 2004; Jaenicke and Decker 2004; Decker 2005), but recently major advances have been achieved in our laboratories in Mainz/Germany that are summarized in the present report. This encompasses (1) extension of the cooperative Nested MWC model to include explicitly effector binding properties and proof that hemocyanins can occur in different conformations (Decker, Hellmann); (2) functional adaptation of crustacean hemocyanins to different environmental temperatures (Hellmann, Decker); (3) the molecular basis of the activation of hemocyanins and phenoloxidases (Jaenicke, Decker); (4) evolution of molluscan hemocyanins as deduced from cDNA and genomic sequences (Lieb, Markl); and (5) 3D cryo-electron microscopy of hemocyanins to determine the contact areas between substructures for understanding the transmission of the conformational signals (Meissner, Markl).
There has also been substantial progress—with many contributions from researchers in Mainz/Germany—concerning DNA sequencing and evolution of arthropod hemocyanins (Voit et al. 2000; Burmester 2001; Ballweber et al. 2002; Burmester 2002; Averdam et al. 2003) and electron microscopy of the disassembly/reassembly of molluscan hemocyanins (for references, see Harris and Markl 1999; Harris et al. 2004), but this will not be further addressed here.
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Cooperative and allosteric oxygen-binding behavior of hemocyanins |
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While the cooperative oxygen-binding behavior of arthropod hemocyanins has been well described in terms of the oxygen-binding properties, the characteristics of allosteric effector binding was not investigated in detail so far. Particularly, in crustaceans several compounds of low molecular weight modulate hemocyanin oxygen binding, resulting in lower or higher oxygen affinities of the respiratory protein (Bridges 2001
). This influence of effectors on oxygen-binding behavior is often quantified via the shift of p50 of the corresponding oxygen-binding curve. The dependence of p50 on effector concentration can be approximated as follows (Wyman and Gill 1990):
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Here, Wn,eff and Wo,eff denote the so-called binding polynomials for the effector in the fully oxygenated and fully unligated states ("n" and "o", respectively) (Wyman and Gill 1990). In the most simple case, the Monod–Wyman–Changeux (MWC) model (Monod et al. 1965) is applicable which requires two conformations (Fig. 2). Then, at sufficiently high cooperativity, the oxy-state corresponds to one conformational state (R) and the deoxy-state to another (T). In this most simple case, if m identical binding sites for a particular effector exists for state R, one would set Wn,eff = WR,eff = (1 + Keff,R[eff])m, with Keff,R being the binding affinity of the effector for the R-state and [eff] the concentration of free effector. For binding to the T-state a corresponding expression would be used. In this case, intrinsic effector-binding properties (stoichiometry and affinity for the T- and the R-state) can be deduced from an analysis of the shift in p50 without further analysis of the data on oxygen binding in terms of a specific model. Examples can be found elsewhere (Johnson et al. 1987; Sanna et al. 2004).
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Arthropod hemocyanins larger than hexamers cannot be described by the simple MWC model but are in very good agreement with a hierarchical extension of it: the Nested MWC model (Fig. 2) (Decker et al. 1986
, 1988; Decker and Sterner 1990; Sterner and Decker 1990; Makino and Ohnaka 1993; Dainese et al. 1998; Molon et al. 2000). Here, hierarchies in allosteric equilibria are assigned to obvious structural hierarchies (Robert et al. 1987). The Nested MWC model is characterized by four conformations (tT, tR, rR, and rT). Effectors such as urate, lactate, protons, water/glycine and Ca2+ shift the equilibria between these conformations (Brenowitz et al. 1983; Mangum 1983; Makino 1986; Morris and Bridges 1986; Johnson et al. 1988; Lallier and Truchot 1989a, 1989b; Decker and Sterner 1990; Giardina et al. 1992; Nies et al. 1992; Zeis et al. 1992; Sterner and Decker 1994; Hellmann 2004; Hellmann et al. 2003; Sanna et al. 2004; Menze et al. 2005). Thus, the oxy-state and the deoxy-state are expected to consist of a mixture of at least two conformations and an approach based on equation 1 is not possible since the binding polynomials are very complex. In this case the modulation of the allosteric equilibrium constants by the effector has to be exploited. The shift in the allosteric equilibrium between two conformations due to presence of an effector can be related to the ratio of the corresponding binding polynomials Q ß for the conformations described ( ß = tT, tR, rR, and rT). In the following equations the allosteric equilibrium constants of the Nested MWC model (lR, lT, and 
) in presence of an effector (with index "eff") relative to the corresponding allosteric equilibrium constants in absence of the effector are given.
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For definition of the expressions in the brackets see caption for Fig. 2. Due to the complexity of the model, however, analysis of data on oxygen binding alone does not always lead to unambiguous results for the parameters describing oxygen and effector binding, even if binding data at different effector concentration are analyzed simultaneously. Fortunately, for the effector urate the binding behavior has been determined directly, either by dialysis (Nies et al. 1992) or more accurately by isothermal titration calorimetry (Menze et al. 2000, 2001, 2005; Hellmann et al. 2001). Although these data alone, again, are not sufficient to extract all relevant binding parameters, the combined analysis of data on oxygen and effector binding allowed us to propose a model for the modulation of the 2 x 6mer hemocyanin from the lobster Homarus vulgaris (Menze et al. 2005). Here, even nonallosteric urate-binding sites were identified, which were also found for the hemocyanin from the freshwater crayfish Astacus leptodactylus (Hellmann et al. 2001). The metabolic effectors urate and lactate usually modulate the functional properties of crustacean hemocyanin, albeit not in all cases. Interestingly, recently a crustacean hemocyanin war reported hemocyanin was also reported to be modulated by L-lactate (Paoli et al. 2007). In the case of the hemocyanin from H. vulgaris, the experiments also revealed that hemocyanin is most likely needed, mainly under hypoxic conditions, employing urate as an effector ensuring sufficient oxygen delivery (Menze et al. 2005). Also for protons as effectors, additional information about release and uptake of protons upon oxygen binding allowed us to estimate the pK-values for protons binding for the different conformational states (Sterner and Decker 1994; Hellmann 2004).
It has been proposed that an hierarchical extension of the MWC model, such as the Nested MWC model, allows effectors to act at different levels of structural hierarchies. Indeed, examples can be found where this is exactly observed experimentally. A very clear, albeit not physiologically relevant, example is the effect of Tris on the oxygen-binding properties of tarantula 4 x 6mer hemocyanin (Sterner et al. 1994). Here, only one of the three allosteric equilibrium constants of the Nested MWC model is shifted by the effector. For protons as effectors, typically all three allosteric equilibrium constants are shifted, but L usually much less so than the two allosteric equilibrium constants relating to the small allosteric units (Decker and Sterner 1990; Dainese et al. 1998; Molon et al. 2000). The effect of hydration/osmotic pressure also seems to operate mainly at the level of the small allosteric unit (Hellmann et al. 2003). In the case of urate binding to hemocyanin from H. vulgaris, no strong preference for modulation of one of the hierarchical levels has been observed. Rather, preferential binding to conformation rR occurred (Menze et al. 2005). Similar results were found for the dye Neutral Red (Sterner and Decker 1990) and the binding of urate and L-lactate to hemocyanin from H. americanus (unpublished results). Since the Nested MWC model offers much more flexibility in terms of selective population of specific states, this might be considered as a driving force for the development of this regulatory mechanism in hemocyanin function.
Cooperativity requires the existence of conformations characterized by different affinities and structures. For several hemocyanins, conformations in the oxy-state and deoxy-state have been detected physically by low-resolution small-angle X-ray, neutron scattering and fluorescence spectroscopy which provided important support for the Nested MWC model. (Decker et al. 1996; Hartmann et al. 2001, 2004; Hartmann and Decker 2004; Erker et al. 2004a, 2004b, 2005a, 2005b). While all these experiments were performed on ensembles of hemocyanins, we also tried to follow the oxygenation of individual hemocyanin molecules by monitoring intrinsic tryptophans (Erker et al. 2005a, 2005b; Lippitz et al. 2002) which required functional attachment of hemocyanin to surfaces (Erker et al. 2006).
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Functional adaptation of crustacean hemocyanins to different temperatures |
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Arthropods can live at very different environmental temperatures, for example in hot deserts or in the cold depths of the sea, and many of them, notably most chelicerates and the higher crustaceans, depend for survival upon hemocyanin as the oxygen-carrying respiratory protein of the hemolymph. We investigated the oxygen-binding behavior of such hemocyanins at different temperatures and tried to deduce from these data which oxygen binding parameters might be optimized. To this end, we compared the O2-binding behavior of hemocyanins from two arachnids (a tarantula and a scorpion) and from five different crustacean species living at different typical environmental temperatures. We wanted to know which oxygen binding parameters are influenced by the temperature to what exent in order to understand which properties are selected for optimisation. In such a study, it has to be taken into account that a change in temperature in these poikilothermic animals inevitably leads to a change in hemolymph pH. The extent of this pH change is similar to the pH-change of Tris-buffer: an increase of temperature by 10°C results in a decrease of pH of about 0.3 units. Thus, we compared the temperature dependence of oxygen binding in two types of data sets: in one set, the pH was adjusted to a given value at each temperature; in another set, the temperature-induced pH shift was not corrected, reflecting the behavior in vivo. This very complex analysis is still underway, but all binding curves have been amenable to analysis according to the MWC model (Monod et al. 1965
for single hexamers) and the Nested MWC-model (Robert et al. 1987 for oligo-hexamers).
The following picture emerges from the few species studied so far: hemocyanin from species which are exposed, in their natural habitat, to considerable temperature change exhibit a strong temperature dependence of oxygen affinity and cooperativity as quantified by p50 and n50, respectively; maximum cooperativity is kept at the observed temperature, usually the mean temperature of the natural habitat (Fig. 3). In hemocyanins studied so far, at low temperatures cooperativity increases with pH, while at higher temperatures the cooperativity seems to be rather independent of the pH value. A second clear correlation could be found between the typical temperature of the habitat and the p50 (Fig. 4). For example hemocyanin from the stone crab Paralithodes camtschatica (a 2 x 6mer; Molon et al. 2000) living at a depth of about 100 m off the eastern cost of Siberia at 1–5°C releases bound oxygen at these low temperatures. In contrast, hemocyanin of the tarantula Eurypelma californicum (a 4 x 6mer; Markl and Decker 1992), living in the southwestern deserts of the US, at high temperatures such as 30°C still has more than 90% of the binding sites occupied with oxygen. Under these conditions the hemocyanin of the stone crab is already completely unloaded. So, both oxygen affinity and cooperativity are optimized for a certain temperature range, and the oxygen-transport capacity of hemocyanin is adapted to the environmental temperature of the animals.
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In another but preliminary work on the influence of temperature on the binding behaviour reported at the Bonn meeting, Pott and Grieshaber (2006
) investigated the binding of urate and caffeine by the hemocyanin of the European lobster, Homarus vulgaris, at different temperatures under normoxic and hypoxic conditions using isothermal titration calorimetry. These data were also interpretable by the Nested MWC model, including non-allosteric binding sites based on a former analysis by Menze et al. (2005). |
Activation of hemocyanins to phenoloxidase, and the catalytic mechanism |
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Hemocyanins have type-3 copper centers similar to those of the phenoloxidases, despite their different functions (van Holde et al. 2001
). Hemocyanins serve as oxygen carriers in mollusks and arthropods, whereas phenoloxidases (comprising tyrosinases and catecholoxidases) are ubiquitous essential enzymes, occurring in all organisms studied (Solomon et al. 1994; Decker and Tuczek 2000; Jaenicke and Decker 2004; Terwilliger and Ryan, 2006). Phenoloxidases catalyze the initial step in melanin synthesis and are necessary for multiple biological functions, such as browning, wound healing, primary immune defense and sclerotization. Tyrosinase catalyzes two steps, the hydroxylation of monophenols to O-diphenols and the oxidation to O-chinons, without releasing any intermediates (Decker et al. 2006). Catecholoxidases catalyze only the second reaction. Thus, although these two enzymes share a very similar active site, they catalyze different reactions; the reason for this phenomenon is still unclear, since the crystal structures of a plant catecholoxidase (Klabunde et al. 1998) and a bacterial tyrosinase (Matoba et al. 2006) are very similar and give no obvious hint for the differences in function. However, recent results indicate that hemocyanins, notably those from the chelicerates Eurypelma califormicum and Pandinus imperator, can also develop tyrosinase activity after activation (Decker and Rimke 1998) (Nillius et al. unpublished data). In addition, these arthropod tyrosinases are hexamers similar in structure to resembled hexameric arthropod hemocyanins (Jaenicke and Decker 2003).
On this molecular basis, we developed a hypothesis for the activation of hemocyanins and phenoloxidases, to be described below. Moreover, we will focus on the differential catalysis mechanisms. Hemocyanins as well as phenoloxidases can be activated by limited proteolysis, or by inorganic as well as by organic compounds (Jaenicke and Decker 2004). Most useful is an assay with SDS, allowing formation of SDS micelles at the applied SDS concentration. Under these conditions, arthropod and molluscan hemocyanins show a very similar movement of a flexible structural domain (Decker and Tuczek 2000), thus opening an entrance for bulky phenolic compounds to the active site (Fig. 5). This seems also to be the case with phenoloxidases, which can be grouped as a-phenoloxidases (from "arthropod related") and m-phenoloxidases (from "molluscan related") (Jaenicke and Decker 2004; Decker and Jaenicke 2004). In contrast, the active site of tyrosinase becomes freely accessible after release of the so-called caddie protein, which is necessary for the import of the copper atoms to the active site (Matoba et al. 2006; Claus and Decker 2006).
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The opened entrance allows only one specific orientation for the phenols to approach the active site and therefore enables only one chemical reaction (Decker et al. 2000
). The orientation of the substrate will then be defined by its 
– interaction with the histidine located at the entrance to the coordinating CuB. From here the phenol moves forward so that the hydroxyl group is fixed by a free axial coordination point of the CuA site (Decker et al. 2006). Then, one atom of the dioxygen molecule attacks the phenol ring in the ortho position, forming a hydroxyl group which results in an O-diphenol being immediately oxidized. The other oxygen combines with protons to form a water molecule. Thus, a hypothesis for the basic mechanism of tyrosinase catalysis can now be proposed, but it still remains unclear why two different enzymes exist, tyrosinase and catecholoxidase, because the tyrosinase can catalyze both reactions.
How is phenoloxidase-activated hemocyanin used in vivo ? Typically the hemolymph concentration of tarantula hemocyanin is very high (usually ca. 50 mg/ml), but also cases with only 5 mg/ml were reported without obvious impact on the viability of the animal. Thus it was suggested that about 5 mg/ml are enough to ensure sufficient oxygen transport, e.g., Paul et al. (1994). Our suggestion is that the high hemocyanin concentration in tarantula hemolymph might play an important role in the innate immunology of these animals by synthesizing the polymer melanin.
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Primary structure and the evolution of molluscan hemocyanins |
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Hemocyanin is most abundant in gastropods, cephalopods, and chitons, and it also occurs in protobranch bivalves. The polypeptides (subunits) of molluscan hemocyanins are extremely large (either ca. 350 or ca. 400 kDa) and therefore are rich in structural/sequence information applicable to phylogenetic studies. Beginning in 1998, a number of these polypeptides from differerent molluscan taxa have been completely sequenced, at both the cDNA and the genomic level (Miller et al. 1998
; Lieb et al. 2000, 2001; 2004 Altenhein et al. 2002; Bergmann et al. 2006, 2007). These results allow reconstruction of a rather robust phylogenetic tree from multiple sequence alignment (Fig. 6). This tree shows that the eight different functional units (FUs) that constitute a molluscan hemocyanin subunit evolved prior to the divergence of this animal phylum into different classes. In several of the studied molluscs, two distinct hemocyanin isoforms have been detected, that according to biochemical observations (Markl et al. 1991; Gebauer et al. 1994) and whole-mount in situ hybridization studies (Streit et al. 2005, 2006) are differentially expressed. In Haliotis tuberculata, we identified the rhogocytes (pore cells) as the site of hemocyanin biosynthesis (Albrecht et al. 2001). According to our estimations of the molecular clock, the gene duplications yielding the two isoforms occurred independently in different taxa, and coincide with molluscan evolutionary hotspots, e.g., specific radiation periods (Bergmann et al. 2006, 2007).
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Moreover, our data provide strong evidence that the eight different FUs (termed FU-a to FU-h) that constitute the subunit evolved ca. 740 million years ago (late Precambrian) by three subsequent events of gene duplication and fusion from a precursor protein that contained a single copper active site; we also provided a hypothesis for the stepwise evolution of the native (ca. 4 MDa) decamer from a dipentameric (ca. 600 kDa) ring (Lieb et al. 2000
, 2001). Within the Cephalopoda, the C-terminal FU-h is missing, which is clearly an apomorphy. The three putative gene duplications and fusions were probably facilitated by two bordering introns that are still present as primordial "linker" introns (separating the exons encoding the different FUs) in all studied hemocyanin genes (Lieb et al. 2001, 2004; Altenhein et al. 2002; Bergmann et al. 2006, 2007). During further evolution, additional taxon-specific "internal" introns were inserted occasionally and split the coding sequences of individual FUs into two or more exons (Fig. 7). There are also indications of an excessive loss of such internal introns within certain taxa.
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Apart from such phylogenetic considerations, the elucidated sequences are the basis of studies on structure–function and expression, and for our current attempts at recombinant expression. Moreover, they enable construction of molecular models of the individual FUs in a hybrid approach that combines data from X-ray crystallography and 3D-cryoelectron microscopy (described in the next section).
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3D cryo-electron microscopy of arthropod and molluscan hemocyanins |
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Hemocyanin sequences from different molluscan classes are now available and crystal structures are known for two different FU types (FU-e and FU-g) (Cuff et al. 1998
; Perbandt et al. 2003), and we have available a wealth of electron microscopical and biochemical data on the quaternary structure (Siezen and van Bruggen 1974; van Holde and Miller 1995; Harris and Markl 1999). Nevertheless, many details of the architecture of the decamer—the basic oligomeric assembly of native molluscan hemocyanin—are still unclear. The hemocyanin decamer consists of a wall made from 10 copies of the subunit segment a-b-c-d-e-f, and an internal collar complex. In cephalopods such as Octopus and Nautilus, the collar is built from ten copies of FU-g (in Sepia it is different and will not be addressed further here). In gastropods, chitons and the few hemocyanin-containing bivalves, the collar complex additionally contains 10 copies of FU-h. In both versions, however, details of the collar structure, as well as the topology of the wall segment a-b-c-d-e-f, remain obscure. To approach this, we recently performed 3D reconstructions from cryo-electron micrographs of the hemocyanin molecules from the gastropod Haliotis tuberculata (Fig. 8A) and the cephalopod Nautilus pompilius (Fig. 8B), and achieved a resolution of 11Å in both cases (Meissner et al. 2007). Analysis and comparison of these 3D reconstructions revealed that the wall architecture of both hemocyanins is very similar; it shows three tiers, with 20 functional units each that encircle the cylinder wall. Our new data confirm and extend the results from previous lower-resolution structures (Lambert et al. 1994; Mouche et al. 1999, 2003; Meissner et al. 2000). Moreover, in the 11 Å reconstructions, six types of wall FUs were individually structurally discernable and found to be strikingly similar in both hemocyanins (Fig. 8). Also, the internal collar complex of the decamers showed superior resolution, and specific differences were detected between the two hemocyanins. The five FU-g pairs of the central collar (in both hemocyanins) and the five FU-h pairs of the peripheral collar (only in Haliotis hemocyanin) are now clearly defined, as well as their connections to the wall and to each other. The FU-g pairs are attached to the wall through a previously unknown structural element that we termed the anchor (Meissner et al. 2007). Crystallization of FU-h is presently in progress, and a 4 Å structure is already available (unpublished data). After the Bonn meeting, we considerably improved the present 3D reconstruction of Nautilus hemocyanin and have currently achieved a resolution of 9 Å. This reconstruction allows a convincing fit of molecular models of the individual FUs into the cryo-EM structure, and has ultimately revealed what we now consider to be the correct pathway of the FUs within the folded subunit (Gatsogiannis et al., manuscript in preparation).
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The 8 x 6mer hemocyanin of horseshoe crabs is the largest arthropod hemocyanin found in nature (3.5 MDa) and it exhibits a highly cooperative oxygen binding. The 48 subunits of this hemocyanin are arranged as eight hexamers (1 x 6mers) that form the native 8 x 6mer, in a nested hierarchy of 2 x 6mers and 4 x 6mers. There are eight distinct subunit types (for historical reasons termed I, IIA, II, IIIA, IIIB, IV, V, and VI) in a known stoichiometry and topology (Boisset et al. 1988
). Prior to our recent study on the 8 x 6mer of Limulus polyphemus (Martin et al. 2007), several crystal structures and a 10-Å cryo-EM structure of the basic 1 x 6mer has been published (Volbeda and Hol 1989; Hazes et al. 1993; Magnus et al. 1994; Meissner et al. 2003). From the 8 x 6mer (or any other arthropod hemocyanin oligo-hexamer), however, only a 40-Å structure existed previously (Taveau et al. 1997). By 3D cryo-electron microscopy and single-particle analysis, we ultimately obtained a 10-Å structure of the Limulus hemocyanin 8 x 6mer, and by DNA sequencing, molecular modelling and rigid-body fitting we developed a molecular model (Martin et al. 2007). On this basis (Fig. 9), the structural parameters of the 8 x 6mer have been firmly established, and a total of 46 molecular interfaces between the eight hexamers clearly defined. They group as 11 types of interface. Among the amino acids localized at these interfaces we detected various histidine clusters that might transfer allosteric signals between the different levels of the nested hierarchy (Martin et al. 2007).
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Future perspectives |
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Presently, we continue to use 3D-electron microscopy to study hemocyanin molecules in their fully oxygenated and deoxygenated state, in an attempt to unravel possible allosteric structural changes caused by movements of amino acids at the inter-subunit interfaces. This will provide a perfect test system for the Nested MWC model. Concerning further primary structure analyses, apart from phylogenetic considerations we use the obtained hemocyanin sequences and cDNA clones for our current attempts at recombinant expression. Is phenoloxidase-activated hemocyanin really needed in vivo ? Melanin is involved in wound healing, defense against invaders, and sclerotization of the exoskeleton after molting, most likely together with hemocyanin and related proteins (Terwilliger et al. 2005
). Experiments aimed at monitoring this process in vivo are in progress, such as the detection of 18F-dopa derivatives within living animals, using positron emission tomography (PET). Is it a common principle that oxygen affinity and cooperativity of hemocyanin are optimized for a certain temperature range, and that the oxygen-transport capacity of hemocyanin is adapted to the environmental temperature of the animals? And if so, what are the consequences in the context of global warming?
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Acknowledgments |
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Supported by grants from the DFG (Ma843/5; Li998/1; De414/8; De414/12, Ha2844/3, He2620/6, SFB625 B5 (HD, JM), GK1043), the BMBF, EU D21 COST, the biosyn company (Fellbach, Germany), and the Stiftung Innovation of Rheinland-Pfalz. We thank Prof. Dr J. Robin Harris for critical reading of the manuscript.
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Footnotes |
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This paper summarizes one of the 22 symposia that constituted the "First International Congress of Respiratory Biology" held August 14–16, 2006, in Bonn, Germany.
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