Hemocyanins and Invertebrate Evolution

million years ago. The circulatory transport of oxygen is essential for efficient aerobic metabolism in most animals. A variety of proteins has evolved to facilitate this process. Most familiar are the hemoglobins, with representatives in almost every phylum (1Hardison R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5619-5675Crossref PubMed Scopus (291) Google Scholar). However, hemoglobins are not the unique oxygen carriers; there exist two less well known protein classes: (a) the hemerythrins, non-heme iron proteins found in a few invertebrates (2Kurtz D.M. Adv. Comp. Environ. Physiol. 1992; 13: 151-171Crossref Google Scholar) and (b) hemocyanins, binuclear type 3 copper proteins utilized by many arthropods and molluscs (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar). Because they are found freely dissolved in the blood, hemocyanins are easily purified and were the first proteins to be physically characterized as defined, multisubunit structures (4Svedberg T. Chirnoaga E. J. Am. Chem. Soc. 1928; 50: 1399-1411Crossref Scopus (9) Google Scholar). However, their great size (some are over 107 Da) and subunit complexity hindered further study for many years. Only in the last decade have we gained a detailed understanding of how these giant molecules are constructed and how their structures might have evolved from simpler molecules. This, in turn, has provided intriguing clues concerning the evolution of invertebrates and their respiratory functions. Connecting the molecular biology of hemocyanins to invertebrate evolution is the object of this review. The copper-containing oxygen transport proteins of both molluscs and arthropods were originally given the same name: hemocyanin. This was justified by similarities in the mode of oxygen binding. In both phyla, the oxygen-binding site involves a pair of copper atoms, which are in the Cu(I) state in the deoxy form but become Cu(II) upon oxygenation, binding the oxygen as O22−. This change accounts for the blue color developed upon oxygenation. Indeed, recent structural analysis has shown that the O22−-binding sites of molluscan and arthropod hemocyanins are very similar, both in the coordination of copper via histidine ligands and the way in which oxygen is bound (5Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonaventura J. Hol W.G. Proteins. 1994; 19: 302-309Crossref PubMed Scopus (389) Google Scholar, 6Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (341) Google Scholar) (Fig.1). In contrast to these similarities molluscan and arthropod hemocyanins are profoundly different in molecular structure at all levels. This can be seen immediately in the differences in quaternary structure schematically depicted in Fig.2. Because of these differences, it has now become customary to consider the molluscan and arthropod hemocyanins as different proteins (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 7van Holde K.E. Miller K.I. Lang W.H. Adv. Comp. Environ. Physiol. 1992; 13: 251-300Google Scholar, 8Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). In the following section we briefly summarize what is now known concerning the structures of these proteins and show that the nature of their relationship may be more subtle than previously thought.Figure 2Representative hemocyanin structures. A, quaternary structure of an arthropod hemocyanin, the 4-hexamer (24 subunits) hemocyanin from the spider Eurypelma californicum (10Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The seven different subunit types are colored differently. B, tertiary structure of an arthropod hemocyanin subunit from L. polyphemus (35Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (319) Google Scholar). The three domains I (green), II (red), and III (purple) are colored differently. C,quaternary structure of a representative molluscan hemocyanin, that of the abalone, Haliotis tuberculata. This consists of 20 polypeptide chains, each containing 8 functional units and therefore 8 binding sites. The functional units are divided (6/2) between the cylindrical wall and an internal collar (36Meissner U. Dube P. Harris J.R. Stark H. Markl J. J. Mol. Biol. 2000; 298: 21-34Crossref PubMed Scopus (52) Google Scholar) (courtesy of Dr. U. Meissner). D, tertiary structure of a molluscan hemocyanin functional unit, the C-terminal unit from the Octopus dofleini hemocyanin (6Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (341) Google Scholar). The two domains are colored differently. Quaternary structures are from high resolution electron microscopy, and subunit structures are from x-ray diffraction studies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Arthropod hemocyanins are built as multiples of hexamers, each hexamer made of monomers of about 75 kDa. An example of a four-hexamer structure is shown in Fig. 2 A. Sequence analysis shows that a given arthropod hemocyanin may contain several variants of the common monomer sequence (see for example Refs. 9Durstewitz G. Terwilliger N. Mol. Biol. Evol. 1997; 14: 266-276Crossref PubMed Scopus (46) Google Scholar and 10Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), each variant occupying a specific position in the whole molecule. The combination of chain variants determines the level to which hexamer association can occur (11Markl J. Biol. Bull. 1986; 171: 90-115Crossref Google Scholar). The sequences are sufficiently similar that all arthropod hemocyanin subunits probably have a tertiary structure similar to the example shown in Fig. 2 B. Each subunit is organized into three domains; the second, highly helical domain carries the active site copper pair. Each copper is ligated by three histidine residues, as shown in Fig. 1, and lies within a 4α-helix bundle reminiscent of hemerythrin or perhaps even the globin fold (12Volbeda A. Hol W.G.J. J. Mol. Biol. 1989; 206: 531-546Crossref PubMed Scopus (79) Google Scholar). Molluscan hemocyanins are built on an entirely different plan, as Fig.2 C shows. The polypeptide chains are very large, about 350–450 kDa each, and each consists of 7 or 8 globular “functional units” connected by linker peptide strands (13Miller K.I. Cuff M.E. Lang W.E. Varga-Weisz P. Field K.G. van Holde K.E. J. Mol. Biol. 1998; 278: 827-842Crossref PubMed Scopus (93) Google Scholar, 14Lieb B. Altenheim B. Markl J. J. Biol. Chem. 2000; 275: 5675-5681Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). In the blood of cephalopod molluscs, like squids or octopi, the circulating hemocyanin exists as decamers of these large subunits, forming hollow cylindrical arrays with 5- or 10-fold axial symmetry. In some other molluscs (chiefly gastropods) dimers or even higher oligomers of these decamers can be found (see Fig. 2 C). Such molecules are truly immense; the structure shown in Fig. 2 C has a molecular mass of about 9 × 106 Da and contains 160 oxygen-binding sites! Sequence analysis (13Miller K.I. Cuff M.E. Lang W.E. Varga-Weisz P. Field K.G. van Holde K.E. J. Mol. Biol. 1998; 278: 827-842Crossref PubMed Scopus (93) Google Scholar, 14Lieb B. Altenheim B. Markl J. J. Biol. Chem. 2000; 275: 5675-5681Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) has revealed that the functional units within a molluscan hemocyanin monomer are quite similar, with 40–50% identity. The tertiary structure of one such functional unit has been determined (6Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (341) Google Scholar) and is depicted in Fig. 2 D. Note that the arthropod subunit and the molluscan functional unit have a quite different tertiary structure. The molluscan unit is smaller (about 50 kDa) than the arthropod subunit and consists of only two domains, an N-terminal highly helical domain carrying the O2 site and a C-terminal domain that is largely β-sheet. Clearly, at most levels, molluscan and arthropod hemocyanins appear to be quite different proteins. Nevertheless, a closer comparison of molluscan functional units with arthropod hemocyanin subunits raises intriguing questions. Although there appears to be very little overall similarity in amino acid sequences, we do detect in the copper-binding regions what appear to be meaningful local similarities. In each case there are two well separated copper-binding regions: that nearer the N terminus is called the “A” site and that nearer the C terminus the “B” site. Fig.3 depicts sequences in the A and B regions of representative arthropod and molluscan hemocyanins and several related binuclear copper proteins. What sequence similarity exists between these proteins is concentrated in these regions. Moreover, x-ray diffraction studies have shown that octopus hemocyanin (6Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (341) Google Scholar), two arthropod hemocyanins (5Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonaventura J. Hol W.G. Proteins. 1994; 19: 302-309Crossref PubMed Scopus (389) Google Scholar, 12Volbeda A. Hol W.G.J. J. Mol. Biol. 1989; 206: 531-546Crossref PubMed Scopus (79) Google Scholar), and catechol oxidase (15Klabunde T. Eicken C. Sacchettini J.C. Krebs B. Nat. Struct. Biol. 1998; 5: 1084-1090Crossref PubMed Scopus (756) Google Scholar) exhibit remarkable similarity in the spatial arrangement of the six histidine ligands that hold the copper atoms. Fig.4 shows that a very particular arrangement of reactive groups forming the active site is necessary to bind dioxygen as is done by type 3 copper proteins. This conservation lies in the three-dimensional structure rather than in the linear sequences. In fact, the sequences determining the similar structures are quite different in the two phyla. This is particularly clear in the A site, where the molluscan and arthropod hemocyanins have a somewhat different sequence arrangement of the three copper-binding histidines. It is often said that the A and B sites in arthropod hemocyanins are very similar, but aside from the location of the binding histidines, this is not so at least insofar as sequence is concerned (Fig. 3). The overall arrangement of the copper-binding histidines in the A site differs in molluscs mainly in the change in sequence position of the second of these three residues (Fig. 3). The only wholly conserved positions in this whole group of proteins are histidines 1 and 3, and the phenylalanine lying four residues upstream from histidine 3. Considering all of these observations, we must conclude that it is an oversimplification to state that molluscan and arthropod hemocyanins are unrelated. However, the relationship must be distant.Figure 4A superposition of the binding site regions in the arthropod (Limulus polyphemus(purple)) subunit and the molluscan (Octopus dofleini (blue) functional unit. The A site is at the left. Note the close juxtaposition of structure on the B site and of both coppers and all ligating histidines (taken from Ref. 6Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (341) Google Scholar with permission).View Large Image Figure ViewerDownload Hi-res image Download (PPT) A clue to the origins of the hemocyanins can be seen in Fig. 3 and from examination of the much larger body of data of which this represents a small sample. Both classes of hemocyanins appear to be related to proteins exhibiting phenoloxidase activity (16Decker H. Terwilliger N. J. Exp. Biol. 2000; 203: 1777-1782PubMed Google Scholar, 17Decker H. Tuczek F. Trends Biochem. Sci. 2000; 25: 392-397Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The arthropod hemocyanins exhibit some sequence similarity to arthropod phenol oxidases (see Refs. 18Aspan A. Huang F.S. Cerenius L. Söderhäll K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (230) Google Scholar and 19Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar, for example), whereas the molluscan hemocyanins resemble more closely that group of enzymes known as tyrosinases (20van Gelder C.W.G. Flurkey W.H. Wichers H.J. Phytochemistry. 1997; 45: 1309-1323Crossref PubMed Scopus (406) Google Scholar) and catechol oxidases (15Klabunde T. Eicken C. Sacchettini J.C. Krebs B. Nat. Struct. Biol. 1998; 5: 1084-1090Crossref PubMed Scopus (756) Google Scholar). These differential sequence similarities are especially clear in the region of the active site, as shown in Fig. 3. Furthermore, structural comparison of sweet potato catechol oxidase and the hemocyanin-binding domain from octopus reveals a specific similarity with respect to an unusual Cys-His thioether bridge, which holds one of the copper-binding histidines in the proper orientation. The fact that both kinds of hemocyanins have weak phenol oxidase activity further supports the idea of close affinity (16Decker H. Terwilliger N. J. Exp. Biol. 2000; 203: 1777-1782PubMed Google Scholar, 17Decker H. Tuczek F. Trends Biochem. Sci. 2000; 25: 392-397Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 21Salvato B. Jori G. Piazzese A. Ghiretti F. Beltramini M. Lerch K. Life Sci. Rep. Suppl. 1983; 1: 313-317Google Scholar, 22Salvato B. Santamaria M. Beltramini M. Alzuet G. Casella L. Biochemistry. 1998; 37: 14065-14077Crossref PubMed Scopus (89) Google Scholar). Both tyrosinases and phenol oxidases are widely distributed and ancient. If hemocyanins did evolve from phenoloxidases and tyrosinases the substrate binding capacity of the enzyme must have been inhibited without hindering O2binding. This seems to have been accomplished by the addition of residues blocking the active site to large substrates (17Decker H. Tuczek F. Trends Biochem. Sci. 2000; 25: 392-397Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 23Decker H. Dillinger R. Tuczek F. Angew. Chem. Int. Ed. Engl. 2000; 39: 1591-1595Crossref PubMed Scopus (180) Google Scholar, 24Decker H. Rimke T. J. Biol. Chem. 1998; 273: 25889-25892Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Indeed, just such a difference has been noted by comparing the crystal structure of sweet potato catechol oxidase with that of molluscan hemocyanin (15Klabunde T. Eicken C. Sacchettini J.C. Krebs B. Nat. Struct. Biol. 1998; 5: 1084-1090Crossref PubMed Scopus (756) Google Scholar). If the precursor to hemocyanins was a cytoplasmic enzyme, some mechanism had to be provided to permit transport into the circulatory system. Leader sequences are found on molluscan hemocyanins (25Lieb, B., Altenheim, B., Markl, J., Vincent, A., van Olden, E., van Holde, K., and Miller, K. (2001) Proc. Natl. Acad. Sci. U. S. A., in press.Google Scholar). Arthropod hemocyanins are exported into the hemolymph either by leader sequences or by cell lysis (8Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). However, providing for export is not sufficient; a low molecular weight monomeric protein dissolved in the hemolymph is not efficient for oxygen transport. If its concentration in the hemolymph is high enough to transport a significant amount of oxygen, it will yield an unbearably high osmotic pressure. This problem can be avoided if the subunits aggregate and/or polymerize to yield giant molecules. For example, octopus hemocyanin exists in the hemolymph at a concentration of about 100 mg/ml, yet because of its great molecular mass gives the same osmotic pressure contribution as about 2 mg/ml vertebrate hemoglobin would if it were free in the blood. The evolutionary solution found for this problem differed in the arthropods and molluscs. The former developed subunits that non-covalently associated; the latter appear to have used gene duplication (25Lieb, B., Altenheim, B., Markl, J., Vincent, A., van Olden, E., van Holde, K., and Miller, K. (2001) Proc. Natl. Acad. Sci. U. S. A., in press.Google Scholar) to generate long, multiunit chains, which then further polymerize. These strategies, which generated large macromolecular structures with many binding sites, not only decreased the osmotic pressure but also provided a new versatility in facing the problem of loading oxygen with high affinity at the respiratory interface and unloading the oxygen where it is This is accomplished by a to molecules with binding sites. hemocyanins the found in with of more than 9 R. J. Comp. Physiol. B. Scopus Google Scholar). how molluscan and arthropod hemocyanins have from but similar enzymes with phenol oxidase sequence similarities between these enzymes the in an even more common It has often been that the difference in the A site between molluscan and arthropod hemocyanins different for these two classes of proteins (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 7van Holde K.E. Miller K.I. Lang W.H. Adv. Comp. Environ. Physiol. 1992; 13: 251-300Google Scholar, 8Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). In it was that the arthropod copper-binding region from a duplication of a B site, whereas the molluscan copper-binding domain from a between two one carrying an A site type of structure and the other a B site (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 7van Holde K.E. Miller K.I. Lang W.H. Adv. Comp. Environ. Physiol. 1992; 13: 251-300Google Scholar, H. Terwilliger N. J. Exp. Biol. 2000; 203: 1777-1782PubMed Google Scholar). However, closer examination of Fig. 3 as to so an is as shown that the A and B sites are very similar in the of histidines and and and the conserved phenylalanine is in the same in both sites. the truly residues, only histidine 2 has between what we type and type copper proteins. the for the sequence differences is that all type 3 copper proteins have evolved from a binuclear which from duplication of a copper site. The of these into and then evolution to yield the quite different sequences found (see Fig. The between the molluscan hemocyanins sequence and tyrosinases as with that between arthropod hemocyanins and phenol oxidases that the molluscan is more (see of active site even evolutionary that the protein is by A well example is found in and the T. Proteins. Ed. H. Scholar). Because arthropod and molluscan hemocyanins to have evolved from somewhat different we consider their origins as the between the the and the which the are now as (see Refs. A. G. N. B. R. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar and S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus (180) Google In either the evolution of a functional hemocyanin must have been quite as by the for the of either arthropod subunits within one (10Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google or molluscan functional units (13Miller K.I. Cuff M.E. Lang W.E. Varga-Weisz P. Field K.G. van Holde K.E. J. Mol. Biol. 1998; 278: 827-842Crossref PubMed Scopus (93) Google Scholar, 14Lieb B. Altenheim B. Markl J. J. Biol. Chem. 2000; 275: 5675-5681Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). circulating hemocyanins they evolved into like the structures. to the in evolutionary at which the molluscan and arthropod hemocyanins first appear as functional may an concerning the of of the phyla. for the of this over a from the of the S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus (180) Google Scholar) to J. Mol. Evol. 1998; 47: PubMed Scopus Google Scholar, 1996; Scopus Google Scholar). The the first of but of are well known into the It seems that some of these to arthropods and molluscs S. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus (180) Google Scholar, 1997; Scopus Google Scholar). In this what do the hemocyanin sequence data as such as the of from or arthropods from other it is to of hemocyanin subunit functional sequences to the at which the units For the data to a of the subunits the first functional arthropod at about (10Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, T. Mol. Biol. Evol. PubMed Scopus Google Scholar). This is a with which the more for evolution would be The molluscan hemocyanin on the other yield an that the of molluscan functional units between about and B. Altenheim B. Markl J. J. Biol. Chem. 2000; 275: 5675-5681Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). If this is for the the of these we can a very for the evolution of the in the evolution of the was A. 1996; PubMed Scopus Google Scholar). these the oxygen by must have been to many A variety of enzymes were evolved to oxygen by carrying For this two were iron and In the case of a type 3 copper in which two coppers oxygen as a (5Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonaventura J. Hol W.G. Proteins. 1994; 19: 302-309Crossref PubMed Scopus (389) Google Scholar, 6Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (341) Google Scholar). The tyrosinases and other phenol oxidases must an of binuclear copper the of higher phyla. It is that these all from a protein was to from the new the the to oxygen were close to at the A. 1996; PubMed Scopus Google Scholar), and aerobic metabolism had been well An in as well as the of made for oxygen a circulating oxygen transport protein essential to the of aerobic that such transport proteins developed in several hemoglobins from from and the two kinds of hemocyanins from two different classes of phenol The of such transport from different that it the of the phyla. This in that the hemocyanin evolution in the molluscan is by both the of molluscan functional unit and by the evolutionary between molluscan hemocyanins and their (Fig. 3). A is shown in Fig. the such must of be with more sequence data on these and other invertebrate proteins are