maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 151
tain precise and accurate masses of native molecules [5, 6].
To obtain the masses of each constituent, gel electrophoresis
has been widely used but the current method of choice is
mass spectrometry under denaturing conditions. By associating
data concerning both the native protein and its subunits,
new insights into the quaternary structure and the fine tuning
of its physiological functions can be achieved.
1. INVERTEBRATE OXYGEN BINDING PROTEINS:
THE EXAMPLE OF GIANT HEXAGONAL BILAYER
HEMOGLOBIN AND HEMOCYANIN
Oxygen binding proteins occur in all phyla but reach a
high diversity in metazoans [2-4]. They exhibit a high diversity
of localization and structure. They are found in tissues
and in coelomic or vascular fluids. They can be stored in
cells (intracellular pigments) or freely dissolved in the internal
fluids (extracellular pigments). They are metalloproteins
in which either iron (hemoglobin, hemerythrin) or copper
(hemocyanin) reversibly bind O 2 .
In hemoglobins, one iron atom is bound to the tetrapyrrole
ring to form the heme group. The heme is linked to a
polypeptide which presents a typical globin-fold [4]. They
have been notably observed in annelids, arthropods and molluscs.
They range from single chains (e.g. myoglobin, cytoglobin,
leghemoglobin) found in bacteria, algae, protozoa
and plants to large, multisubunit, multidomain complexes
found in nematodes, molluscs and crustaceans, and the giant
annelid hemoglobins comprised of globin and nonglobin
subunits [4]. In hemocyanins, two copper atoms are bound to
histidine residues of the polypeptide chain; the resulting
“blue” proteins are present in molluscs and arthropods.
In this review, we will focus on the analytical structure of
two classes of multimeric respiratory pigments: the annelid
extracellular hexagonal bilayer hemoglobins (HBL-Hb) and
crustacean hemocyanins (Hc). These pigments exhibit high
complexation levels and the wide range of living environments
of the concerned animal phyla allows for different
functional properties among species. In addition, the recently
proposed use of Arenicola marina Hb (AmHb) as a model
for a potential blood substitute [7, 8] and the use of arthropod
Hc as a model for nested allostery further support the
interest of structural investigation of these complexes [9-12].
1.1. The Hexagonal Bilayer Hemoglobin (HBL-Hb) of
Annelids
The annelid phylum comprises over 8000 species subdivided
into terrestrial oligochaetes, aquatic leeches, and marine
polychaetes. Annelids are present in a wide variety of
biotopes, from aerated soil (Lumbricus terrestris) to challenging
hypoxic or toxic environments (intertidal muds for
Arenicola marina; deep-sea hydrothermal vents for Alvinella
pompejana). Most of them have extracellular or intracellular
hemoglobins (Hbs) and sometimes both [13, 14]. However,
in many annelids, oxygen transport relies on giant extracellular
respiratory proteins (~3.6 x 10 6 Da), known as either
erythrocruorins or hexagonal bilayer hemoglobins (HBL-
Hbs). Such large complexes offer a number of important
advantages as oxygen transport vehicles: HBL-Hbs are readily
retained in the vascular system as freely dissolved entities,
each complex possesses large oxygen binding capacity
and subunits can be arranged to permit cooperative oxygen
binding and additional regulatory features enhancing oxygen
transport. The HBL-Hbs are heteromultimeric complexes
consisting of globins (14-18 kDa) and nonglobin, linker
chains (23-28 kDa) in an approximate 2:1 ratio, and represent
a summit of complexity for heme proteins binding O 2
reversibly. Chlorocruorins (Chl) are respiratory pigments
structurally similar to HBL-Hb except for a different side
group in the tetrapyrrole ring. Their absorption spectrum is
thus modified so that the oxygenated form is green rather
than red. They are found in some annelid families (Sabellidae,
Serpulidae, Chlorohaemidae, Amphateridae [4]).
The HBL-Hbs of the polychaete A. marina and the oligochaete
L. terrestris were among the first proteins investigated
by ultracentrifugation [15] and electron microscopy
[16]. They exhibit a highly symmetric hexagonal bilayer
appearance (HBL), with dimensions of about 20 nm x 30 nm
[17]. The first biochemical studies by Svedberg and collaborators
yielded sedimentation constant for numerous erythrocruorins,
ranging from 54.1 S to 61.5 S, and also produced a
first estimation of the mass for A. marina and L. terrestris
hemoglobins of about 2.8 MDa [15, 18].
HBL-Hbs exhibit some original and remarkable properties,
such as an acidic isoelectric point, a high resistance to
auto-oxidation and self-assembling properties, which could
be explained by the selection of robust structures for oxygen
transport as a freely dissolved molecule in the blood. The
high aggregation degree also permits a broad range of cooperativity
(from n 50 = 0.91 in Amphitrite ornata [19] to n 50 =
6.5 in Megascolides australis [20]). A large affinity range is
also observed between species (P 50 = 0.2 Torr for Alvinella
pompejana Hb at 20°C; P 50 = 80 Torr for Spirographis spallanzanii
Chl at 20°C [21]).
As the global structure detected by transmission electron
microscopy (TEM) seems quite uniform in annelids from
different biotopes, the understanding of the inner structural
organization of these respiratory complexes is of crucial interest
to point out potential specific molecular adaptations to
the environmental conditions.
The determination of the precise composition and structure
of these complexes has been a major issue in invertebrate
Hb biochemistry for a long time. Determination of a
model called for knowledge of the complex mass, the
subunit masses and stoichiometry and the spatial arrangement
of the chains. Several models were proposed to account
for the hexagonal bilayer structure observed for all extracellular
giant hemoglobins and the relationship between globin
and linker subunits. Determinations of native masses sometimes
led to different results as is the case for L. terrestris
[22, 23]. Availability of high resolution methods for determination
of native and denatured masses was a keystone in
the resolution of parts of the controversies in this field.
1.2. Arthropod Hemocyanin (Hc)
Hemocyanin is the respiratory pigment responsible for
dioxygen transport in many arthropods and molluscs. It is a
polypeptide complex of high molecular mass, ranging from
450 kDa (crustaceans) to about 10 MDa (gastropods). They
share a seemingly similar active site involving two Cu atoms
72