From the Department of Biochemistry and Biophysics,
Oregon State University, Corvallis, Oregon 97331-7305 and
Institute for Molecular Biophysics, University of Mainz,
D55128 Mainz, Germany
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 (1). 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 (2) and (b)
hemocyanins, binuclear type 3 copper proteins utilized by many
arthropods and molluscs (3). 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 (4).
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
INTRODUCTION
TOP
INTRODUCTION
Hemocyanin Structure: One...
From What Did Hemocyanins...
The Emergence of Hemocyanin...
A Common Ancestor for...
When Did Hemocyanins Originate?
REFERENCES
Hemocyanin Structure: One Protein or Two?
TOP
INTRODUCTION
Hemocyanin Structure: One...
From What Did Hemocyanins...
The Emergence of Hemocyanin...
A Common Ancestor for...
When Did Hemocyanins Originate?
REFERENCES
. 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 (5, 6) (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 (3, 7, 8). 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.
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Fig. 1.
The oxygen-binding site common to type 3 copper proteins. The specific structure shown here is taken from
an x-ray diffraction study of Limulus polyphemus hemocyanin
(5). Virtually identical structures have been observed in several other
type 3 copper proteins. Coppers are blue and oxygens are
red.
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Fig. 2.
Representative hemocyanin structures.
A, quaternary structure of an arthropod hemocyanin, the
4-hexamer (24 subunits) hemocyanin from the spider Eurypelma
californicum (10). The seven different subunit types are colored
differently. B, tertiary structure of an arthropod
hemocyanin subunit from L. polyphemus (35). 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 (36) (courtesy of Dr. U. Meissner). D, tertiary structure of a molluscan hemocyanin
functional unit, the C-terminal unit from the Octopus
dofleini hemocyanin (6). The two domains are colored differently.
Quaternary structures are from high resolution electron microscopy, and
subunit structures are from x-ray diffraction studies.
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. 2A. Sequence analysis shows that
a given arthropod hemocyanin may contain several variants of the common
monomer sequence (see for example Refs. 9 and 10), each variant
occupying a specific position in the whole molecule. The combination of
chain variants determines the level to which hexamer association can
occur (11). The sequences are sufficiently similar that all arthropod
hemocyanin subunits probably have a tertiary structure similar to the
example shown in Fig. 2B. 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 4-helix bundle reminiscent of
hemerythrin or perhaps even the globin fold (12).
Molluscan hemocyanins are built on an entirely different plan, as Fig.
2C 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 (13, 14). 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. 2C). Such molecules are truly
immense; the structure shown in Fig. 2C has a molecular mass
of about 9 × 106 Da and contains 160 oxygen-binding
sites! Sequence analysis (13, 14) 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 (6) and is depicted in Fig. 2D. 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 -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
(6), two arthropod hemocyanins (5, 12), and catechol oxidase (15) 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.
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From What Did Hemocyanins Evolve? |
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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 (16, 17). The
arthropod hemocyanins exhibit some sequence similarity to arthropod
phenol oxidases (see Refs. 18 and 19, for example), whereas the
molluscan hemocyanins resemble more closely that group of enzymes known
as tyrosinases (20) and catechol oxidases (15). 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 (16, 17, 21, 22). 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 O2
binding. This seems to have been accomplished by the addition of
residues blocking the active site to large substrates (17, 23, 24).
Indeed, just such a difference has been noted by comparing the crystal
structure of sweet potato catechol oxidase with that of molluscan
hemocyanin (15).
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The Emergence of Hemocyanin Function |
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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 (25). Arthropod hemocyanins are exported into the hemolymph either by leader sequences or by cell lysis (8).
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 (25) 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 needed. This is accomplished by cooperative binding, a behavior confined to molecules with multiple binding sites. Some hemocyanins possess the highest cooperativity found in nature, with Hill coefficients of more than 9 (26).
Thus, current evidence indicates how molluscan and arthropod
hemocyanins could have arisen independently from distinct but similar
enzymes with phenol oxidase activities. Faint sequence similarities
between these enzymes (mainly near the O2-binding site)
suggest in turn an even more remote common ancestor.
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A Common Ancestor for all Type 3 Copper Proteins? |
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It has often been suggested that the difference in the A site
between molluscan and arthropod hemocyanins indicates fundamentally different ancestries for these two classes of proteins (3, 7, 8). In
particular, it was proposed that the arthropod copper-binding region
arose from a simple duplication of a primordial B site, whereas the
molluscan copper-binding domain resulted from a fusion between two
genes, one carrying an A site type of structure and the other a B site
(3, 7, 16). However, closer examination of Fig. 3 casts doubt as to
whether so complex an explanation is necessary. Alignment as shown here
indicates that the A and B sites are very similar in the placement of
histidines 1B and 1A, and 3B and 3A. Also, the conserved phenylalanine
residue is in exactly the same registry in both sites. Of the truly
critical residues, only histidine 2 has shifted significantly between
what we call type 3a and type 3b copper proteins. Perhaps the simplest explanation for the present sequence differences is that all type 3 copper proteins have evolved from a binuclear predecessor, which itself
arose from duplication of a B-like copper site. The branches of these
proteins, segregating into proto-molluscan and proto-arthropodan lines
then underwent independent evolution to yield the quite different
sequences found today (see Fig. 5). The
greater dissimilarity between the molluscan hemocyanins sequence and
tyrosinases as compared with that between arthropod hemocyanins and
insect phenol oxidases suggests that the molluscan
hemocyanin-tyrosinase split is more ancient (see below). Conservation
of active site geometry, even through evolutionary changes that greatly
modify the surrounding protein framework, is by no means unknown. A
well recognized example is found in subtilisin and the mammalian serine
proteases (27).
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When Did Hemocyanins Originate? |
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Because arthropod and molluscan hemocyanins seem to have evolved independently from somewhat different (but related) proteins, we should consider their origins as separate events, occurring sometime after the split between the Lophotrochozoa (which includes the molluscs) and the Ecdysozoa (of which the arthropoda are now recognized as members (see Refs. 28 and 29)). In either case, the evolution of a fully functional hemocyanin must have been quite rapid, as judged by the starlike branching patterns for the diversification of either arthropod subunits (even within one species (10)) or molluscan functional units (13, 14). That is, once circulating hemocyanins appeared they rapidly evolved into something like the present structures. Attempts to define the points in evolutionary time at which the molluscan and arthropod hemocyanins first appear as functional entities encounter (and may contribute to) an ongoing debate concerning the time of divergence of the major phyla. Estimates for the onset of this divergence range over half a billion years, from the beginning of the Cambrian, 550 MYA1 (29) to 800-1000 MYA (30, 31). The early Cambrian demonstrates the first profusion of shelly fossils, but fossils of softer bodied metazoans are well known back into the Vendian (550-650 MYA). It seems likely that some of these creatures, although difficult to classify, represented primitive arthropods and molluscs (29, 32). In this context, what do the hemocyanin sequence data tell us? By using as calibration dates events such as the divergence of cephalopods from gastropods or chelicerate arthropods from other arthropods, it is possible to use comparisons of hemocyanin subunit (or functional unit) sequences to estimate the time at which the multiple units arose. For arthropods, the data point to a rapid divergence of the chelicerate subunits (and presumably the first fully functional arthropod hemocyanin) at about 550-600 MYA (10, 33). This is a date with which the more conservative models for metazoan evolution would be comfortable. The molluscan hemocyanin data, on the other hand, yield an apparently surprising result, that the diversification of molluscan functional units occurred between about 700 and 800 MYA (14). If correct, this is indeed strong evidence for the "phyla-early" models.
On the basis of these results, we can propose a very tentative scenario
for the evolution of the hemocyanins. Very early in the evolution of
life, the environment was anaerobic (34). Under these circumstances,
the oxygen production produced by photosynthesis must have been toxic
to many creatures. A variety of enzymes were evolved to neutralize
oxygen by carrying out oxidation reactions. For this two metal ions
were used, iron and copper. In the case of copper, a type 3 copper
center evolved, in which two coppers reversibly bond oxygen as a
peroxide (5, 6). The resulting tyrosinases and other phenol oxidases
must represent an extremely ancient class of binuclear copper proteins,
predating the emergence of higher metazoan phyla. It is possible that
these all originated from a single protein whose function was to
protect primitive organisms from the new toxin, oxygen. By the time the
major metazoan phyla began to emerge (700-800 MYA?) oxygen levels were
close to those at the present (34), and aerobic metabolism had been well established. An increase in animal size, as well as the
development of impermeable integuments, made simple diffusion
inadequate for oxygen supply. Therefore, a circulating oxygen transport
protein became essential to utilize the advantages of aerobic
metabolism. We postulate that such transport proteins developed in
several independent ways, hemoglobins from myoglobins, hemerythrins
from myohemerythrins, and the two kinds of hemocyanins from two
different classes of phenol oxidases. The development of such diverse
transport systems, from different roots, strongly suggests that it
occurred after the divergence of the major phyla. This in
turn argues that the phylogenetic divergence occurred before 750 MYA.
That hemocyanin evolution occurred earlier in the molluscan line is
indicated by both the earlier dating of molluscan functional unit
divergence and by the apparent greater evolutionary distance between
molluscan hemocyanins and their tyrosinase cousins (Fig. 3). A
reasonable scenario is shown in Fig. 5. At the present, such
speculations must of course be regarded with caution. Much more
sequence data on these and other invertebrate proteins are urgently needed.
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FOOTNOTES |
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* This minireview will be reprinted in the 2001 Minireview Compendium, which will be available in December, 2001.
§ Supported by a research grant from the National Science Foundation.
¶ To whom correspondence should be addressed. Tel.: 541-737-2264; Fax: 541-737-0481; E-mail: millerk@ucs.orst.edu.
** Supported by the Deutsche Forschungsgemeinschaft.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.R100010200
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ABBREVIATIONS |
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The abbreviation used is: MYA, million years ago..
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