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BLOOD PIGMENTS
Haemocyanin
Hemocyanins are respiratory proteins in the form of metalloproteins containing two copper atoms that
reversibly bind a single oxygen molecule (O2). Oxygenation causes a color change between the colorless Cu(I)
deoxygenated form and the blue Cu(II) oxygenated form. Hemocyanins carry oxygen in the blood of most
molluscs, and some arthropods, including the horseshoe crab, Limulus polymephus. They are second only to
hemoglobin in biological popularity of use in oxygen transport.Unlike hemoglobin, hemocyanins are not
bound to blood cells, but suspended in the hemolymph.
Although the respiratory function of hemocyanin is similar to that of hemoglobin, there are a significant
number of differences in its molecular structure and mechanism. Whereas hemoglobin carries its iron atoms in
porphyrin rings (heme groups), the copper atoms of hemocyanin are bound as prosthetic groups coordinated
by histidine residues. Species using hemocyanin for oxygen transportation are commonly crustaceans living in
cold environments with low oxygen pressure. Under these circumstances hemoglobin oxygen transportation is
less efficient than hemocyanin oxygen transportation.
Most hemocyanins bind with oxygen non-cooperatively and are roughly one-fourth as efficient as hemoglobin
at transporting oxygen per amount of blood. Hemoglobin binds oxygen cooperatively due to steric
conformation changes in the protein complex, which increases hemoglobin's affinity for oxygen when partially
oxygenated. In some hemocyanins of horseshoe crabs and some other species of arthropods, cooperative
binding is observed, with Hill coefficients of 1.6 - 3.0. Hill constants vary depending on species and laboratory
measurement settings. Hemoglobin, for comparison, has a Hill coefficient of usually 2.8 - 3.0. In these cases of
cooperative binding hemocyanin was arranged in protein sub-complexes of 6 subunits (hexamer) each with
one oxygen binding site; binding of oxygen on one unit in the complex would increase the affinity of the
neighboring units. Each hexamer complex was arranged together to form a larger complex of dozens of
hexamers. In one study, cooperative binding was found to be dependent on hexamers being arranged together
in the larger complex, suggesting cooperative binding between hexamers. Hemocyanin oxygen-binding profile
is also affected by dissolve-salt ion levels and pH.
Hemocyanin is made of many individual subunit proteins, each of which contains two copper atoms and can
bind one oxygen molecule (O2). Each subunit weighs about 75 kilodaltons (kDa). Subunits may be arranged in
dimers or hexamers depending on species, the dimer or hexamer complex is likewise arranged in chains or
clusters in weights exceeding 1500 kDa. The subunits are usually homogeneous, or heterogeneous with two
variant subunit types. Because of the large size of hemocyanin, it is usually found free-floating in the blood,
unlike hemoglobin, which must be contained in cells because its small size would lead it to clog and damage
blood-filtering organs such as the kidneys. This free-floating nature can allow for increased hemocyanin
density over hemoglobin and increased oxygen carrying capacity. On the other hand, free-floating hemocyanin
can increase viscosity and increase the energy expenditure needed to pump blood.
Catalytic Activity
It is interesting to compare hemocyanin to the phenol oxidases (e.g. tyrosinase), homologous enzymes sharing
its type 3 Cu active site coordination. Hemocyanin also exhibits phenol oxidase activity, but with slowed
kinetics from greater steric bulk at the active site. Partial denaturation actually improves hemocyanin’s phenol
oxidase activity by providing greater access to the active site.
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Structure
Oxygen binding mode with respect to copper centers
Hemerythrin
Hemerythrin is an oligomeric protein responsible for oxygen (O2) transportation in the marine invertebrate
phyla of sipunculids, priapulids, brachiopods, and in a single annelid worm, magelona. Recently, hemerythrin
was discovered in methanotrophic bacterium Methylococcus capsulatus. Myohemerythrin is a monomeric O2binding protein found in the muscles of marine invertebrates. Hemerythrin and myohemerythrin are
essentially colorless when deoxygenated, but turn a violet-pink in the oxygenated state.
Hemerythrin does not, as the name might suggest, contain a heme. The names of the blood oxygen
transporters hemoglobin, hemocyanin, hemerythrin and vanabins, do not refer to the heme group (only found
in globins), but are derived from the Greek word for blood.
As it turns out, the oxygen binding site is a binuclear iron center. The iron atoms are coordinated to the protein
through the carboxylate side chains of a glutamate and aspartate and five histidine residues. Hemerythrin and
myohemerythrin are often described according to oxidation and ligation states of the iron center:
Fe2+—OH—Fe2+
Fe2+—OH—Fe3+
Fe3+—O—Fe3+—OOHFe3+—OH—Fe3+—
deoxy (reduced)
semi-met
oxy (oxidized)
(any other ligand)
met (oxidized)
The uptake of O2 by hemerythrin is accompanied by two-electron oxidation of the reduced binuclear center to
produce bound peroxide (OOH-). The chemistry of O2 binding is summarized in scheme:
Deoxyhemerythrin contains two high-spin ferrous ions bridged by hydroxyl group. One iron is
hexacoordinate and another is pentacoordinate. The bridging hydroxyl serves as the proton donor for peroxide
after O2 binding, resulting in the formation of a single oxygen atom (μ-oxo) bridge in oxy- and
methemerythrin. O2 binds to the pentacoordinate Fe2+ at the vacant coordination site. Then electrons are
transferred from the ferrous ions to generate the binuclear ferric (Fe3+,Fe3+) center with bound peroxide.
Hemerythrin typically exists as a homooctamer or heterooctamer composed of α- and β-type subunits of 13-14
kDa each, although some species have dimeric, trimeric and tetrameric hemerythrins. Each subunit has a fourα-helix fold binding a binuclear iron center. Because of its size hemerythrin is usually found in cells or
"corpuscles" in the blood rather than free floating.
Unlike hemoglobin, most hemerythrins lack cooperative binding to oxygen making it roughly 1/4 as efficient
as hemoglobin. In some brachiopods though, hemerythrin shows cooperative binding of O2. Cooperative
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binding is achieved by cooperation between subunits: when one subunit becomes oxygenated it increases the
affinity for oxygen of the other subunits in the complex.
Hemerythrin affinity for carbon monoxide (CO) is actually lower than its affinity for O2 (unlike hemoglobin
which has a very high affinity for CO) making hemerythrin immune to CO poisoning. This is due to
hemerythrin binding mechanism (shown above) which cannot bind CO with stability
Chlorocruorin
Chlorocruorin is an oxygen carrying blood protein of many annelids, it is noted for appearing green when
deoxygenated (as opposed to dark red like hemoglobin), though it is light red when oxygenated. Its structure
is very similar to erythrocruorin (which is likewise very similar to multiple subunits of myoglobin) and it
contains many 16-17kDa myoglobin-like subunits arranged in a giant complex of over a hundred subunits
with interlinking proteins as well with a total weight exceeding 3600kDa. Because of its giant macromolecule
structure, chlorocruorin is free floating in blood. The only significant difference between chlorocruorin and
erythrocruorin is that chlorocruorin carries an abnormal heme group structure.
Erythrocruorin is a large oxygen-carrying protein, whose molecular mass is greater than 3.5 million Daltons. It
is related to the similar Chlorocruorin. It is found in many annelids.