Respiratory adaptations in a deep-sea orbiniid polychaete from Gulf of Mexico brine pool NR-1: metabolic rates and hemoglobin structure/function relationships
1 Department of Biology, 208 Mueller Lab, Pennsylvania State University,
University Park, PA 16802, USA
2 Station Biologique de Roscoff, BP74, CNRS-UPMC-INSU, 29682 Roscoff cedex,
France
3 Center for Respiratory Adaptation (CRA), Department of Zoophysiology,
University of Aarhus, 8000 Aarhus C, Denmark
4 Micromass Ltd, Tudor Road, Altrincham, Cheshire WA14 5RZ, UK
5 Institute for Storage Ring Facilities Aarhus (ISA), University of
Aarhus, 8000 Aarhus C, Denmark
* Present address: Department of Biology, 208 Mueller Lab, Pennsylvania State
University, University Park, PA 16802, USA
(e-mail: hourdez{at}psu.edu )
Accepted 20 March 2002
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Summary |
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Key words: hypoxia, anoxia-tolerance, sulphide-tolerance, functional properties, cold seep, oxygen consumption, physiology, haemoglobin, orbiniid, polychaete, Methanoaricia dendrobranchiata
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Introduction |
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At Brine Pool NR1, the mussel bed rings the edge of the hypersaline pool on
unconsolidated sediments saturated with brine. The oxygen concentration in
water sampled between the mussels averaged 39 µmol l-1 and was
often undetectable by gas chromatography (<5 µmol l-1;
Smith et al., 2000). Although
no sulfide could be detected in the brine pool itself, millimolar levels are
present in the brine-saturated sediments under the outer edge of the mussel
bed (Smith et al., 2000
). The
combination of sulfidic and hypoxic conditions presents a major respiratory
challenge for active metazoans (Somero et
al., 1989
; Grieshaber and
Völkel, 1998
). Similar conditions characterize the
microhabitats of many hydrothermal vent communities
(Johnson et al., 1988
; Fisher
et al., 1988a
,
b
;
Sarrazin and Juniper,
1999
).
Several studies on hydrothermal vent polychaetes have shown larger
mass-specific gill surface areas and shorter diffusion distances between the
environment and the body fluids than in related genera from other environments
(Jouin and Gaill, 1990;
Hourdez and Jouin-Toulmond,
1998
). The gills of M. dendrobranchiata are also
hypertrophied compared with those of other orbiniids and resemble those of the
vent polychaetes (Hourdez et al.,
2001
). This characteristic and the short diffusion distance across
the gill epithelia have been interpreted as adaptations for life in a hypoxic
environment (Jouin and Gaill,
1990
; Jouin-Toulmond et al.,
1996
; Hourdez and
Jouin-Toulmond, 1998
; Hourdez
et al., 2001
).
In addition to these anatomical adaptations, the respiratory pigments of
the vent polychaetes and vestimentiferan species studied to date have very
high oxygen affinities and strong Bohr effects that may facilitate oxygen
uploading in the gills at low oxygen tensions as well as its release in the
tissues (Arp et al., 1990;
Toulmond et al., 1990
;
Hourdez et al., 1999
). M.
dendrobranchiata contains an extracellular hemoglobin in its blood
vessels (Hourdez et al.,
2001
). In annelids, such extracellular hemoglobins are usually
giant hexagonal bilayer (HBL) molecules, made up of 12 large subunits
(dodecamers) composed of globin chains. These dodecamers are held together by
polypeptide chains known as linkers (for a review, see
Lamy et al., 1996
).
We studied aspects of the respiratory physiology of M. dendrobranchiata from the cold seeps of the Gulf of Mexico. The relationship between oxygen consumption rate and environmental oxygen concentration was determined together with tolerance of anoxia in both the presence and absence of sulfide. We also determined the concentration of hemoglobin in the worms and describe the molecular and functional properties of the purified molecule. Finally, these findings are discussed in the context of the animal's habitat and behavior.
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Materials and methods |
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Oxygen consumption rates
The oxygen consumption rates of individual worms were determined in
water-jacketed, gas-tight chambers (Fisher
et al., 1985) at 8.5±0.5°C using Clark-type oxygen
probes (Orion 835 dissolved oxygen meter)
(Fig. 1). Oxygen concentrations
in the chamber were recorded every 5 min for 16-17 h. After the experiment,
the worms were rapidly dried with paper tissues and frozen in cryovials for
transport to Pennsylvania State University. To determine the wet mass of the
worm, the cryovial was weighed before and after removing the worm,
Mass-specific oxygen consumption rates were calculated for each 20-min
interval (using five data points) and plotted against the mean oxygen
concentrations for that time interval.
|
Anoxia and sulfide-tolerance
Worms were maintained anaerobically in water containing 0, 0.06 or 1 mmol
l-1 sulfide to determine their tolerance to anoxia under these
conditions. Oxygen-free sea water was obtained by bubbling with nitrogen for
30 min and then transferred to a nitrogen atmosphere in a glove bag in a cold
room at 8 °C, where all subsequent manipulations were conducted. The
absence of detectable oxygen was confirmed with a dissolved oxygen meter
(Orion 835). Preweighed washed crystals of sodium sulfide were then added, and
the sulfide concentrations were measured by gas chromatography
(Childress et al., 1984).
Individual worms were incubated separately in sealed 30 ml glass scintillation
vials secondarily contained in a 500 ml gas-tight container filled with
nitrogen-equilibrated water (to dampen minor temperature fluctuations and
maintain anaerobic conditions). Eleven vials containing one worm each were
incubated anaerobically under each experimental condition, and an additional
22 worms from the same collection were kept under normoxia in individual vials
that were open to the atmosphere (controls). Survival was checked every 12 h
until the ship reached port (7 days).
To evaluate the time corresponding to a 50 % survival rate
(TL50), the following survival curve was fitted to the
data:
![]() | (1) |
Extraction and purification of hemoglobin
Because of the very small blood vessels, it was not possible to sample pure
blood intravascularly. Approximately 75 worms from a single collection were
batch-frozen 1 h after recovery for shipment to the laboratory and hemoglobin
extraction. In the laboratory, the worms were homogenized with 100ml of
extraction buffer (50 mmoll-1 Tris, pH8, 1 mmoll-1 EDTA
and 1 µmoll-1 phenylmethanesulfonic fluoride, PMSF). The
homogenate was centrifuged for 20 min at 20 000 g to remove the
coarse debris. The bright red supernatant was then centrifuged at 100 000
g for 5 h to pellet the hemoglobin. The pellet containing the
hemoglobin was resuspended in 1 ml of the extraction buffer, and the
hemoglobin was purified by fast protein liquid chromatography (FPLC) on a
Superose 6 column (10 mmx300 mm; Pharmacia Inc., 5-5000 kDa separation
range) using 400 mmoll-1 NaCl, 2.95 mmoll-1, KCl, 32
mmoll-1 MgSO4, 11 mmoll-1 CaCl2
and 50 mmoll-1 Hepes at pH 7.0 as the elution buffer. The elution
rate was typically 0.5 ml min-1, and the elutant was monitored at
280 nm. The molecular mass of the collected fractions was estimated using a
sample of Riftia pachyptila blood, which contains four peaks of known
molecular mass (Zal et al.,
1996). The presence of hemoglobin in colored fractions was
verified on the basis of peak absorptions near 414 nm.
Hemoglobin content
To assess the significance of the hemoglobin for oxygen storage, 61 worms
ranging widely in size (0.08-1.95 g) were individually frozen on board ship.
In the laboratory, they were weighed and homogenized individually, using a
ground-glass tissue homogenizer, in 1 mmoll-1 Tris buffer, pH 8,
containing 1 mmoll-1 EDTA and 1 µmoll-1 PMSF to
prevent hydrolysis by proteases. The homogenate was centrifuged at 10 000
g for 15 min to remove cellular debris, and the supernatant was used
for heme concentration measurements after appropriate dilution of the samples
in the extraction buffer. Potassium ferricyanide solution (10
mmoll-1; 10 ml) was added to 2 ml of this diluted solution, which
had been incubated for 5 min at room temperature to oxidize the hemoglobin
completely to methemoglobin. Potassium cyanide (50 mmoll-1; 10 ml)
was then added to obtain the cyan-methemoglobin derivative, which was
quantified on the basis of the absorbance of the solution at 540 nm and the
most appropriate published extinction coefficient of 11.0l mmol-1
cm-1 (Van Assendelft,
1970).
Negative-stain transmission electron microscopy of purified, whole
hemoglobin molecules
Images of negatively stained M. dendrobranchiata hemoglobin
molecules were recorded by transmission electron microscopy to assess their
molecular organization following a method modified from Valentine et al.
(1968). Diluted purified
hemoglobin (3 µl; 0.037 mmoll-1 heme in 100 mmoll-1
Tris buffer at pH 6.85) was applied to a very thin (10-30 nm) carbon foil
supported on a standard 3.05 mm copper electron microscopy grid. After
allowing 1 min for the hemoglobin molecules to adhere to the carbon foil, 6
µl of 2 % (m/v) uranyl acetate solution was applied to stain the molecules.
After 10 s, the grid with the stained sample was blotted with Whatman filter
paper, leaving only a very thin wet layer on the carbon foil, which was
allowed to dry in air. The negatively stained specimens were examined using a
Philips CM120 electron microscope at 100 keV, and images were recorded under
minimum-dose conditions (Kenney et al.,
1995
) at 40 000x magnification.
The transmission electron microscopy images of the negatively stained
hemoglobin molecules were analyzed to reveal the symmetry of their
macromolecular structure. The images were digitized, and individual hemoglobin
molecules were selected and centered by reference free alignment using the
program SPIDER (Frank et al.,
1996). The rotational frequency analysis of SPIDER-processed
images was performed using the Medical Research Council image-processing
programs (Crowther et al.,
1996
), and the rotational power spectra of the images were
calculated.
Electro-spray ionization mass spectrometry
Electro-spray ionization mass spectrometry (ESI-MS) was used under
denaturing conditions to determine the molecular masses of the chains and
covalently bound subunits present in the native hemoglobin and in the 210 kDa
fraction (see Results). Three different treatments were applied to this
hemoglobin sample to analyze its structure, as described previously (Green et
al., 1996,
1999
): (i)
carboxyamidomethylation (Cam) to determine the number of free cysteines, (ii)
reduction with dithiothreitol (DTT) to determine the composition of dimeric
species and the reduced masses of the chains, and (iii) reduction plus Cam
(reduced/Cam), to determine the total number of cysteine residues in each
chain. The DTT reduction was carried out at a concentration of 5
mmoll-1 for 2 min at 20 °C. Briefly, samples were introduced at
a rate of 5 µl min-1 into the electrospray source of a Quattro
LC mass spectrometer (Micromass UK Ltd). They were analyzed at a concentration
of 0.25-0.5 mg ml-1 in 1:1 (by volume) acetonitrile:water
containing 0.2 % formic acid. The scan range was m/z 600-2500, and
the cone voltage (counter electrode to skimmer voltage) ramp was from 30 V at
m/z 600 to 100 V at m/z 2500 (m is mass; z
is charge). Mass scale calibration employed the multiply charged series of
ions from horse heart myoglobin (Mr 16 951.5 Da; Sigma,
M-1882). Data were processed using the Maximum Entropy (MaxEnt)-based software
supplied with the instruments. Molecular masses are based on the atomic masses
of the elements: C=12.011, H=1.00794, N=14.00674, O=15.9994 and S=32.066
(IUPAC).
Oxygen equilibrium curves
Equilibrium curves were obtained with a diffusion chamber apparatus
(Sick and Gersonde, 1969)
modified as described previously (Weber,
1981
). Briefly, small (4 µl) samples of purified hemoglobin
were equilibrated with pure (>99.998 %) N2 and O2 and
mixtures of these gases and air prepared using Wösthoff pumps. The pH of
the hemoglobin solutions was varied by adding 6 µl of 1 moll-1
Bis-Tris Propane buffer of a range of pH values to 100 µl aliquots of the
sample, and water was added to bring the total volume to 120 µl. The pH was
measured in duplicate with a blood gas analyzer (BMS2, Radiometer) on 50 µl
subsamples. Oxygen equilibrium and pH measurements were carried out at 10, 20
and 30 °C (±0.1 °C).
Values of P50 (the PO2
at which the hemoglobin is half-saturated with oxygen) and
n50 (cooperativity at P50) were
derived from linear regression Hill plots of
log[Y/(1-Y)]=f(logP02)
for S (saturation values) of 30-70 %. The Bohr factor () was
calculated as
=
logP50/
pH, and the
apparent heat of oxygenation was calculated as
Hobs=2.303RlogP50/[1/T1)-(1/T2)],
where R is the gas constant and T1 and
T2 are the absolute temperatures. The value thus obtained
(
Hobs) comprises the intrinsic heat of oxygenation
(
Hintr), the heat of solution of O2
(
Hsol, approximately 13 kJ mol-1) and
contributions from oxygenation-linked processes such as the heats of reaction
with protons and other effectors. In the absence of oxygenation-linked binding
of protons (when
=0) and other allosteric effectors,
Hintr=
Hobs-
Hsol
(see Wyman et al., 1977
).
Values reported as means ± standard deviation (S.D.) throughout this manuscript.
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Results |
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A logistic curve was found to describe the data best on the basis of the
2-value:
![]() | (2) |
Separate linear regressions for points below 0.4 mg O2 l-1 and points above 1.5 mg O2 l-1 allowed us to estimate the critical pressure (the intercept of the two lines). For the worm in Fig. 2, the intercept gives a critical pressure Pc of 0.27 mg O2 l-1 (700 Pa).
These measurements were made for seven worms ranging from 0.64 to 1.87 g in
wet mass. For two of these experiments, the oxygen concentration did not fall
below 1 mg l-1. In these cases, we used the linear portion of the
oxygen concentration as a function of time curve
(Fig. 2A) to calculate the
oxygen consumption rate. For the five other experiments, the shape of the
curve was always the same, and the 2-value supported the use
of the above equation for each data set (P<0.01). The
Pc values ranged from 0.25 to 0.38 mg
O2l-1 in the five experiments
(PO2=650-990 Pa) and oxygen consumption ceased
at 0.09-0.11 mg O2l-1
(PO2=230-280 Pa).
Fig. 3 shows the
relationship between respiratory rate and animal wet mass. The oxygen
consumption rate follows a power function trend described by the equation:
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Anoxia and sulfide-tolerance
Fig. 4 shows the percentage
survival as a function of time under normoxia, anoxia, anoxia + 60
µmoll-1 sulfide and anoxia + 1 mmoll-1 sulfide. None
of the normoxic (control) animals died during the experiment, while only a
single worm survived for 7 days under anoxia. The mortality curves are
sigmoidal. Curve-fitting yield TL50 values (time to reach
50 % survival) of 99.1, 119.6 and 133.5h, respectively, for anoxia + 1
mmoll-1 sulfide, anoxia + 60 µmoll-1 sulfide and
anoxia without sulfide. Analysis of covariance (ANCOVA) of the linearized data
indicated that the effect of the treatment was highly significant, and
pairwise comparisons confirmed that all TL50 values were
significantly different (P<0.01).
|
Hemoglobin content
Fig. 5 shows the hemoglobin
content as a function of wet mass for the 61 worms. The masses ranged from
0.008 to 1.95 g and the hemoglobin (Hb) contents from 143 to 373 nmol Hb
g-1 wet mass. The individual mass-specific hemoglobin content
(Q) increases with the size, reaching a plateau at approximately 250
nmol g-1 wet mass. The mean mass-specific hemoglobin content is
237±52 nmol Hb g-1 wet mass (N=61). The following
equation fits the data, with a correlation coefficient r=0.388
(N=61, P<0.01):
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|
Purification of the hemoglobin
The extracellular hemoglobin was purified by FPLC. Absorbances at 280 nm
(which characterize proteins) and 414 nm (the Soret band, which characterizes
hemoglobins) show two hemoglobin fractions with masses of approximately
3.5x106 Da and 210 kDa. The second fraction, which was very
small (elution profile not shown), is referred to as `dodecamers' (D in the
figures) and is considered to be a dissociation product of the first fraction
of whole hemoglobin molecules (W-Hb) (for an example, see
Zal et al., 1996). Support for
this designation is presented below.
Image analysis of transmission electron microscopy pictures of whole
hemoglobin molecules
The transmission electron microscopy pictures of the whole hemoglobin
molecules (Fig. 6A,B) showed a
strong peak at the sixth rotational harmonic, indicating a sixfold rotation
symmetry (Fig. 6C). This
information was used to produce a rotationally Fourier-filtered image. The
result is a typical top (sixfold axis) view of a hexagonal bilayer (HBL)
(Fig. 6D).
|
Structure of the hemoglobin
Under denaturing conditions, ESI-MS of the native hemoglobin revealed three
groups of peaks with molecular masses of approximately 16, 32 and 49 kDa. In
the first group, four peaks can be distinguished: a1, a2, a3 and a4, with
masses of 15 824.8, 15 835.1, 15 864.5 and 16 255.4±1.5 Da and relative
intensities of 0.40:0.34:0.19:0.17, respectively (N=5)
(Fig. 7A;
Table 1). A single peak (Dm)
occurred at 31 972.6±3.0 Da (N=5), while the third group
consisted of four peaks (LD1-LD4) between 48 715.5 and 48 804.9 Da
(Fig. 7, inset in A). Careful
examination of the ESI spectra from several preparations of W-Hb analyzed
under various conditions failed to reveal the presence of components that
could correspond to trimers or tetramers of globin chains. ESI-MS on the 210
kDa fraction revealed peaks corresponding to a1-a4 and Dm only (no peaks of
higher mass were seen).
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|
Under mild reducing conditions (1 and 2 min in the presence of 5 mmol
l-1 DTT at 22 °C) (Fig.
7B), the masses of a1-a3 remained unchanged, while the mass of a4
decreased by 119.5 Da, suggesting that this chain is cysteinylated.
Concomitantly, the intensity of the Dm peak decreased and the group of peaks
around 49 kDa disappeared. In addition, several new peaks appeared: b, c and d
at 15 373.4, 16 600.6 and 17 172.9 Da, respectively (±1.5 Da,
N=6), and L1, L2, L3, L4 and L5 at 24 358.7, 24 379.0, 24 404.2, 26
279.3 and 27 687.1 Da, respectively (±3.0 Da, N=4). These
results imply that Dm is a disulphide-bonded dimer composed of monomers b and
c, since b+c-2H=31 972.0, which is the mass determined for Dm in the unreduced
hemoglobin (Green et al.,
1996). Furthermore, twice the masses of L1, L2 and L3 minus 2H are
within experimental error of the masses of LD1, LD2 and LD4
(Fig. 7B), implying that L1-L3
exist as disulfide-linked dimers in the native hemoglobin. L1-L5 have masses
that are typical of linker chains. However, no peaks were observed in the
native hemoglobin that could correspond to dimers of L4 and L5. Moreover,
while L1-L3 remained present upon reduction, L4 and L5 disappeared, with the
concomitant appearance of several relatively minor peaks in the range 18-23
kDa. Peak d cannot be assigned to a higher-mass component in the W-Hb. It is
possible that it is one of the components in a dimer or higher-order multimer
that was not detected in the denatured W-Hb. It could also correspond to a
fragment of a linker, made more fragile after reduction.
After carboxyamidomethylation (Cam) with iodoacetamide, the masses of a1-a3 remained unchanged, indicating that they contain no free cysteine. However, the mass of Dm increased by 2 Cam units, implying that it contains two free cysteine residues. The total number of cysteine residues in each chain and the reduced masses of the chains were determined from the reduced/Cam hemoglobin, as shown in Table 1 and Fig. 7C. Strong support for LD1-LD4 being dimeric forms of L1-L3 is provided by the reduced/Cam results as follows. L1-L3 have masses typical of linker chains and appear to exist as dimers in the native hemoglobin. The peak at 48 715.5 Da is probably a dimer made of two L1 chains (2L1-22H=48 710.2 Da) and the peak at 48 755.1 Da could be a dimer made of two L2 chains, or one L1 chain and one L3 chain (2L2-22H=48 757.8 Da and L1+L3-22H=48 757.1 Da, respectively). The peak at 48 777.4 Da is probably a dimer of L2 and L3 (L2+L3-22H=48 780.9 Da) and, finally, the peak at 48 804.9 Da is probably a dimer of two L3 chains (2L3-22H=48 804.0 Da). All these calculated masses are well within the experimental error for the measured masses of the peaks.
Functional properties of the hemoglobin
In Fig. 8, the effects of pH
on the affinity (P50) and cooperativity
(n50) of the intact hemoglobin (W-Hb) and dodecamers (D)
at 10, 20 and 30 °C are shown. W-Hb and D exhibit the same oxygen
affinities at pH 7.6 for 10 °C (P50=27.8 Pa), at pH
7.4 for 20 °C (P50=82 Pa) and at pH 7.1 for 30 °C
(P50=271 Pa). At lower pH values, D shows progressively
higher affinities than W-Hb, whereas the reverse applies at higher pH.
|
Both hemoglobins express Bohr effects in the range pH 6.7-7.5. The Bohr
factors in W-Hb vary inversely with temperature =-0.48, -0.44 and -0.35
at 10, 20 and 30 °C, respectively, and exceed those in D (
=-0.11,
-0.13 and -0.11, respectively).
W-Hb and D also differ in cooperativity coefficients, n50. In W-Hb, the coefficient is higher, with a maximum at pH 7.5, and appears to vary inversely with temperature (n50=2.5 at 10 °C, n50=2.0 at 20 °C and n50=1.8 at 30 °C). In D the value of n50 (1.2) is almost independent of pH.
Extended Hill plots of oxygen equilibria of the intact hemoglobin molecules
(W-Hb) at pH 6.77 and 7.60 are shown in
Fig. 9. The allosteric and
other parameters derived from analysis of the equilibria in terms of the
two-state MonodWymanChangeux (MWC) allosteric model (as
described by Weber et al.,
1995) are given in Table
2. The correspondence between the cooperativity value at
half-saturation (n50) and the maximum cooperativity
(nmax) and between P50 and the median
oxygen affinity Pm
(Table 2) reflects symmetrical
oxygen-binding curves and permits the use of P50 values to
analyse allosteric interactions. The control mechanism of the Bohr effect in
this hemoglobin is primarily an increase in the oxygen-binding affinity of the
hemoglobin in the oxygenated R (`relaxed') state (KR) as pH rises,
while the affinity (KT) of the deoxygenated T (`tense') state
remains relatively constant (Table
2). This raises the free energy of cooperativity
(
G) (Table 2)
and the KT/KR ratio with increasing pH. The analyses
indicate the presence of 3-5 interacting oxygen-binding sites (Q) in
the functional subunits of the intact hemoglobin molecules.
|
|
The oxygen affinities of both hemoglobins show strong
temperature-dependence (Figs 8,
10) at pH 6.8 and 7.6. The
temperature effect is smaller in W-Hb than in D
(Hobs=-58 and -66 kJ mol-1 for W-Hb at pH
6.8 and 7.6, respectively, and -77 kJ mol-1 for D at both pH
values).
|
In contrast to the marked effect of 100 mmol l-1
Ca2+, which increases the oxygen affinity of W-Hb
(logP50=0.28)
(Fig. 11), D lacks sensitivity
to Ca2+.
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Discussion |
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Hemoglobin structure
Intact Methanoaricia dendrobranchiata hemoglobin has a native mass
and appearance (as seen in transmission electron microscopy) typical of giant
HBL annelid hemoglobins (for a review, see
Lamy et al., 1996). On the
basis of the masses of its dissociation products, it contains both globin and
linker chains, as expected for HBL hemoglobins (for a review, see
Lamy et al., 1996
). There
appear to be five linkers, although three of them (L1-L3) behave quite
differently from the other two (L4 and L5) when reduced. Linkers L1-L3 possess
11 cysteine residues, 10 involved in intra-chain disulfide bonds and one
involved in a disulfide bond between two linker chains
(Table 1). All the linkers are
dimeric, as in Arenicola marina
(Zal et al., 1997
), and are
stable as monomers under reducing conditions. However, the other two linkers
(L4 and L5), if indeed they are linkers, only appeared as monomers under mild
reducing conditions and were never observed as either monomers or dimers in
the native hemoglobin. Upon further reduction, they disappeared, and a number
of components in the mass range 18-23 kDa were observed; these then
disappeared after a longer reduction time. At this time, monomeric L1-L3 were
still present, suggesting that L4 and L5 are significantly less stable than
L1-L3. Both monomeric (a1-a4) and dimeric (b and c) globins are present in
M. dendrobranchiata hemoglobin. The globin dimers were unexpected
because dimers have previously been considered to be typical of hirudinean
(leech) and vestimentiferan hemoglobins in contrast to all other studied
polychaetes, which possess globin trimers (for a review, see
Lamy et al., 1996
). This is
also the first record of an annelid globin found to be cysteinylated. The
origin of d remains unknown: it is probably involved in higher aggregation
states, not detected by ESI-MS, or corresponds to a fragment of a linker,
probably L4 or L5. A similar situation, where some subunits do not seem to
ionize properly under non-reducing conditions, was encountered in
Eudistylia vancouverii chlorocruorin
(Green et al., 1998
).
Functional properties
Both intact molecules and dodecamers exhibit similar and very high oxygen
affinities but differ in other oxygen-binding characteristics (cooperativity,
Bohr effect, sensitivity to Ca2+ and sensitivity to temperature).
Their affinities are among the highest reported in annelids and probably
represent adaptations to the animal's low-oxygen environment
(Toulmond et al., 1990; Weber,
1978a
,
1980
;
Toulmond, 1992
; Hourdez et
al., 1999
,
2000
;
Weber and Vinogradov, 2001
).
The increase in KR with rising pH
(Fig. 9) is opposite to the
situation for human hemoglobin (in which the Bohr effect is due to variation
in KT; Tyuma et al.,
1973
), but similar to that in the polychaete Arenicola
marina, where it increases the Bohr factor at high oxygen saturation and
favors oxygen saturation of blood entering the relatively alkaline gills
(Weber, 1980
;
Toulmond, 1992
).
The Bohr effect in the intact molecules (W-Hb) is similar to that of most
extracellular hemoglobins (Weber,
1978a) but is greatly reduced in the dodecamer, as previously
reported for other annelids (for a review, see
Lamy et al., 1996
).
In contrast to similar blood Ca2+ concentrations among
vestimentiferans and other annelids studied (7-13 mmol l-1), blood
Mg2+ levels vary widely among species (from 2 to 44 mmol
l-1) (Oglesby,
1978; Sanders and Childress,
1991
) and thus may affect the functional properties of annelid
hemoglobins in vivo. Ca2+ increases the oxygen affinity of
intact M. dendrobranchiata hemoglobin but has no effect on the
dodecamers, suggesting that the effect of group IIA cations such as
Ca2+ and Mg2+ involves the linkers. This agrees with
other studies on HBL hemoglobin and most studies of dodecamers, with the
exception of Lumbricus terrestris hemoglobin dodecamers, which are
sensitive to group IIA cations (for a review, see
Lamy et al., 1996
).
Lumbricus terrestris hemoglobin has been shown to have
Ca2+-binding sites with a variety of affinities that mainly involve
linkers (Kuchumov et al.,
2000
). The low-affinity sites are likely to be involved in the
effects of group IIA cations on the structure and function of hemoglobin
(Kuchumov et al., 2000
).
The cooperativity coefficients of M. dendrobranchiata hemoglobin
are within the range previously reported for HBL hemoglobins
(Weber and Vinogradov, 2001).
The value for the intact molecule (2.5) is approximately twice that of the
dodecamer (1.2), as seen in Lumbricus terrestris hemoglobin
(n
5 and n
2, respectively, at pH 7.2;
Krebs et al., 1996
). The
difference suggests a participation of the linker chains in heterotropic
interactions in the molecule, as in other HBL hemoglobins (for a review, see
Lamy et al., 1996
).
The temperature-sensitivity of the intact hemoglobin (H=-66
kJ mol-1 at pH 7.6) is similar to that of the polychete
Abarenicola clarapedii, another annelid that lives in a fairly
stenothermal environment, but greater than that of Arenicola marina
(
H=-22 kJ mol-1;
Weber, 1972
), which
experiences larger temperature variations in its natural habitat. Given that
dissociation of Bohr protons is endothermic and reduces the net heat liberated
upon oxygenation of the hemes, the lower temperature effect in the whole
molecules than in the dodecamers (Fig.
10) accords with the larger Bohr effect in the former.
Analogously,
Hobs of the intact molecules is lower
at pH 6.8 than at pH 7.6, where the Bohr effect is smaller (see
Fig. 8).
Adaptations to the environment
The high affinity and strong Bohr effect observed in the vascular
hemoglobin probably have adaptive significance in a low-oxygen environment,
favoring both the binding of oxygen at the respiratory surfaces and its
release in the tissues, where pH decreases as a result of the production of
acid excretory products.
The oxygen consumption rates of M. dendrobranchiata are within the
lower end of the normal range of respiratory rates reported for polychaetes
(Childress and Mickel, 1985;
Weber, 1978b
). The worms are
very good oxyregulators and are able to maintain a constant metabolic rate as
environmental concentrations fall to 0.3 mg O2 1-1
(where Po2=870 Pa)
(Fig. 2). At lower partial
pressures, the oxygen consumption rate drops sharply, and oxygen consumption
ceases below 0.1 mg O2 1-1 (Po2=260
Pa).
Strong oxyregulation extending to very low oxygen concentrations is
normally correlated with one or more of the following adaptations: (i) a large
gas-exchange surface, (ii) a short diffusion distance between the environment
and the body fluids to facilitate gas diffusion, (iii) the presence of a
respiratory pigment to increase the oxygen-carrying capacity of the
circulating body fluid(s), (iv) a high oxygen affinity of the respiratory
pigments to increase oxygen loading at low oxygen tension, (v) a high
ventilatory rate to renew the medium as it becomes depleted of oxygen, and
(vi) a high circulatory rate. M. dendrobranchiata have a large gill
surface area and reduced diffusion distances across their respiratory surface
as a result of intra-epidermal blood vessels in the gills
(Hourdez et al., 2001).
Furthermore, the worms are capable of greatly extending their gills when
exposed to hypoxic and anoxic conditions (S. H., personal observation). The
presence of cilia on the surface of the gills
(Hourdez et al., 2001
)
facilitates renewal of the diffusion-limited water layer. These worms do not
ventilate their gills, and no information is available about ciliary movement
under hypoxia. However, mussels from their natural habitat can create strong,
small-scale currents.
M. dendrobranchiata hemoglobin has a very high oxygen affinity
under physiological conditions; an oxygen partial pressure of 100 Pa, which is
far below Pc, saturates the hemoglobin to 96 % at 10 °C in vitro.
However, the oxygen consumption rate of M. dendrobranchiata decreases
when the oxygen partial pressure falls below 870 Pa, probably as a result of
diffusion-limitation driven by the decreased oxygen concentration gradient
across their gills. However, this value (870 Pa) is low, indicating that the
high oxygen affinity partially offsets the lack of ventilatory capability. The
low Pc value reflects a high degree of oxyregulation,
which is similar to that in Riftia pachyptila but slightly greater
than those in the crab Bythograea thermydron and the clam
Calyptogena magnifica, all from deep-sea hydrothermal-vent habitats
(Childress and Fisher, 1992).
Many deep-sea hydrothermal-vent and cold-seep microhabitats are characterized
by low levels of dissolved oxygen, and endemic animals often show similar
capacities to oxyregulate, delaying the onset of anaerobic metabolism.
Sulfide and anoxia-tolerance
Although the high mass-specific gill surface area favors oxygen uptake, it
cannot selectively favor the entry of oxygen without permitting the inward
diffusion of potentially harmful sulfide
(Somero et al., 1989), which
often occurs at substantial concentrations in the mussel beds
(Smith et al., 2000
;
Nix et al., 1995
). Sulfide
further compromises respiration in most animals by inhibiting respiration at
the level of cytochrome c oxidase and hinders binding of oxygen to
most hemoglobins (Somero et al.,
1989
). The sulfide-tolerance under anoxia of these worms compares
with that of Marenzelleria cf. wireni and is the highest
found among the species studied to date
(Fig. 12;
Groenendaal, 1980
;
Schiedeck et al., 1997
;
Gamenick et al., 1998
). The
hemoglobin-bound oxygen store is not enough to sustain aerobic metabolism for
more than 31 min. However, worms faced with prolonged anoxia switch to
anaerobiosis and can survive for days even in the presence of sulfide.
|
Cold seeps, like hydrothermal vents, are habitats characterized by very
high productivity and biomass compared with the generally nutrient-poor deep
sea. The high level of endemism and low species-richness of these communities
suggest that these environments are also physiologically challenging and that
they require specific adaptations. We suggest that the ability of
Methanoaricia dendrobranchiata to colonize their extreme environment
is due at least in part to the adaptations reported here, such as their
capacity to oxyregulate, which is due to the presence of a high-affinity
hemoglobin, and their tolerance to anoxia and sulfide. These adaptations allow
the worm to forage in the sulfidic, organic-rich sediments between and below
the mussels and to exploit a habitat with little or no competition and limited
predation (MacAvoy et al.,
2002).
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Acknowledgments |
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References |
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Arp, A. J., Doyle, M. L., Di Cera, E. and Gill, S. J. (1990). Oxygenation properties of the two co-occurring hemoglobins of the tube-worm Riftia pachyptila. Respir, Physiol. 80,323 -334.[Medline]
Blake, J. (2000). A new genus and species of polychaete worm (Family Orbiniidae) from methane seeps in the Gulf of Mexico, with a review of the systematics and phylogenetic interrelationships of the genera of Orbiniidae. Cah. Biol. Mar. 41,435 -449.
Brooks, J. M., Kennicutt, M. C. I., Fisher, C. R., Macko, S. A., Cole, K., Childress, J. J., Bidigare, R. R. and Vetter, R. D. (1987). Deep-sea hydrocarbon seep communities: evidence for energy and nutritional carbon sources. Science 238,1138 -1142.
Childress, J. J., Arp, A. J. and Fisher, C. R. (1984). Metabolic and blood characteristics of the hydrothermal vent tube-worm Riftia pachyptila. Mar. Biol. 83,109 -124.
Childress, J. J. and Fisher, C. R. (1992). The biology of hydrothermal vent animals: physiology, biochemistry and autotrophic symbioses. Oceanogr. Mar. Biol. Annu. Rev. 30,337 -441.
Childress, J. J., Fisher, C. R., Brooks, J. M., Kennicutt II, M. C., Bidigare, R. and Anderson, A. (1986). A methanotrophic marine molluscan symbiosis: mussels fueled by gas. Science 233,1306 -1308.
Childress, J. J. and Mickel, T. J. (1985). Metabolic rates of animals from hydrothermal vents and other deep-sea habitats. Biol. Soc. Wash. Bull. 6, 249-260.
Crowther, R. A., Henderson, R. and Smith, J. (1996). MRC image processing programs. J. Struct. Biol. 116,9 -16.[Medline]
Fisher, C. R. (1996). Ecophysiology of primary production at deep-sea vents and seeps. In Biosystematics and Ecology Series, vol 11, Deep-Sea and Extreme Shallow-Water Habitats: Affinities and Adaptations (ed. F. Uiblein, J. Ott and M. Stachowtisch), pp. 313-336. Vienna: Austrian Academy of Sciences Press.
Fisher, C. R., Childress, J. J., Arp, A. J., Brooks, J. M., Distel, D., Favuzzi, J. A., Felbeck, H., Fritz, L. W., Hessler, R., Johnson, K. S., Kennicutt II, M. C., Lutz, R. A., Macko, S. A., Newton, A., Powell, M. A., Somero, G. N. and Soto, T. (1988a). Variation in the hydrothermal vent clam, Calyptogena magnifica, at Rose Garden vent on the Galapagos rift. Deep-Sea Res. 35,1811 -1832.
Fisher, C. R., Childress, J. J., Arp, A. J., Brooks, J. M., Distel, D., Favuzzi, J. A., Felbeck, H., Hessler, R., Johnson, K. S., Kennicutt II, M. C., Macko, S. A., Newton, A., Powell, M. A., Somero, G. N. and Soto, T. (1988b). Microhabitat variation in the hydrothermal vent mussel Bathymodiolus thermophilus, at Rose Garden vent on the Galapagos rift. Deep-Sea Res. 35,1769 -1792.
Fisher, C. R., Fitt, W. K. and Trench, R. K. (1985). Photosynthesis and respiration in Tridacna gigas as a function of irradiance and size. Biol. Bull. 169,230 -245.
Frank, J., Radermacher, M., Penczek, P., Zhu, P., Li, Y., Ladjadj, M. and Leith, A. (1996). SPIDER and WEB: processing and visualisation of images in 3D electron microscopy and related fields, J. Struct. Biol. 116,190 -199.[Medline]
Gamenick, I., Vismann, B., Grieshaber, M. K. and Giere, O. (1998). Ecophysiological differentiation of Capitella capitata (Polychaeta). Sibling species from different sulfidic habitats. Mar. Ecol. Prog. Ser. 175,155 -166.
Green, B. N., Hutton, T. and Vinogradov, S. N. (1996). Analysis of complex protein and glycoprotein mixtures by electrospray ionization mass spectrometry with maximum entropy processing. In Methods in Molecular Biology, vol.61 , Protein and Peptide Analysis by Mass Spectrometry (ed. J. R. Chapman), pp. 279-294. Totowa, NJ: Humana Press Inc.[Medline]
Green, B. N., Kuchumov, A. R., Klemm, D. J. and Vinogradov, S. N. (1999). An electrospray ionization mass spectrometric study of the giant, extracellular, hexagonal bilayer hemoglobin of the leech Haemopsis grandis provides a complete enumeration of its subunits. Int. J. Mass. Spec. 188,105 -112.
Green, B. N., Kuchumov, A. R., Walz, D. A., Moens, L. and Vinogradov, S. N. (1998). A hierarchy of disulfide-bonded subunits: the quaternary structure of Eudistylia chlorocruorin. Biochemistry 37,6598 -6605.[Medline]
Grieshaber, M. K. and Völkel, S. (1998). Animal adaptations for tolerance and exploitation of poisonous sulfide. Annu. Rev. Physiol. 60,33 -53.[Medline]
Groenendaal, M. (1980). Tolerance of the lugworm (Arenicola marina) to sulphide. Neth. J. Sea Res. 14,200 -207.
Hourdez, S., Frederick, L. A., Schernecke, A. and Fisher, C. R. (2001). Functional respiratory anatomy of a deep-sea orbiniid polychaete from the Brine Pool NR-1 in the Gulf of Mexico. Invert. Biol. 120,29 -40.
Hourdez, S. and Jouin-Toulmond, C. (1998). Functional anatomy of the respiratory system of Branchipolynoe species (Polychaeta, Polynoidae), commensal with Bathymodiolus species (Bivalvia, Mytilidae) from deep-sea hydrothermal vents. Zoomorphology 118,225 -233.
Hourdez, S., Lallier, F. H., De Cian, M.-C., Green, B. N., Weber, R. E. and Toulmond, A. (2000). The gas transfer system in Alvinella pompejana (Annelida Polychaeta, Terebellida). Functional properties of intracellular and extracellular hemoglobins. Physiol. Biochem. Zool. 73,365 -373.[Medline]
Hourdez, S., Lallier, F. H., Martin-Jézéquel, V., Weber, R. E. and Toulmond, A. (1999). Characterization and functional properties of the extracellular coelomic hemoglobins from the deep-sea hydrothermal vent scaleworm Branchipolynoe symmytilida.Proteins 34,435 -442.[Medline]
Johnson, K. S., Childress, J. J., Hessler, R. R., Sakamoto-Arnold, C. M. and Beehler, C. L. (1988). Chemical and biological interactions in the Rose Garden hydrothermal vent field. Deep-Sea Res. 35,1723 -1744.
Jouin, C. and Gaill, F. (1990). Gills of hydrothermal vent annelids: structure, ultrastructure and functional implications in two alvinellid species. Prog. Oceanogr. 24,59 -69.
Jouin-Toulmond, C., Augustin, D., Desbruyères, D. and Toulmond, A. (1996). The gas transfer system in alvinellids (Annelida, Polychaeta, Terebellida). Anatomy and ultrastructure of the anterior circulatory system and characterization of a coelomic, intracellular, haemoglobin. Cah. Biol. Mar. 37,135 -151.
Kenney, J. M., von Bonsdorff, C.-H., Nassal, M. and Fuller, S. D. (1995). Conformational flexibility and evolutionary conservation in the Hepatitis B virus core structure. Structure 3,1011 -1019.
Kennicutt II, M. C., Brooks, J. M., Bidigare, R. R., Fay, R. R., Wade, T. L. and McDonald, T. J. (1985). Vent-type taxa in a hydrocarbon seep region on the Louisiana Slope. Nature 317,351 -353.
Krebs, A., Kuchumov, A. R., Sharma, P. K., Braswell, E. H.,
Zipper, P., Weber, R. E., Chottard, G. and Vinogradov, S. N.
(1996). Molecular shape, dissociation and oxygen binding of the
dodecamer subunit of Lumbricus terrestris hemoglobin. J.
Biol. Chem. 271,18695
-18704.
Kuchumov, A. R., Loo, J. A. and Vinogradov, S. N. (2000). Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin. J. Prot. Chem. 19,139 -148.[Medline]
Lamy, J. N., Green, B. N., Toulmond, A., Wall, J. S., Weber, R. E. and Vinogradov, S. N. (1996). The giant hexagonal bilayer hemoglobins. Chem. Rev. 96,3113 -3124.[Medline]
MacAvoy, S. E. R., Carney, R. S., Fisher, C. R and Macko, S. A. (2002). Use of chemosynthetic biomass by large, mobile, benthic predators in the Gulf of Mexico. Mar. Ecol. Prog. Ser. (in press).
MacDonald, I. R., Callender, R. W., Burke, R. A., Jr, McDonald, S. J. and Carney, R. S. (1990a). Fine-scale distribution of methanotrophic mussels at a Louisiana cold seep. Prog. Oceanogr. 24,15 -24.
MacDonald, I. R., Guinasso, N. L., Reilly, J. F., Brooks, J. M., Callender, W. R. and Gabrielle, S. G. (1990b). Gulf of Mexico hydrocarbon seep communities. VI. Patterns in community structure and habitat. Geo-Mar. Lett. 10,244 -252.
MacDonald, I. R., Reilly, J. F., Guinasso, N. L., Brooks, J. M., Carney, R. C., Bryant, W. A. and Bright, T. J. (1990c). Chemosynthetic mussels at a brine-filled pockmark in the northern Gulf of Mexico. Science 248,1096 -1099.
Nix, E. R., Fisher, C. R., Vodenichar, J. and Scott, K. M. (1995). Physiological ecology of a mussel with methanotrophic endosymbionts at three hydrocarbon seep sites in the Gulf of Mexico. Mar. Biol. 122,605 -617.
Oglesby, L. C. (1978). Salt and water balance. In Physiology of Annelids (ed. P. J. Mill), pp.555 -658. London, New York, San Francisco: Academic Press.
Sanders, N. K. and Childress, J. J. (1991). The use of single column ion chromatography to measure ion concentrations in invertebrate body fluids. Comp. Physiol. Biochem. 98A,97 -100.
Sarrazin, J. and Juniper, S. K. (1999). Biological characteristics of mosaic communities on hydrothermal edifices at Endeavour Segment, Juan de Fuca Ridge. Mar. Ecol. Prog. Ser. 185,1 -19.
Schiedek, D., Vogan, C., Hardege, J. and Bentley, M. (1997). Marenzelleria cf. wireni (Polychaeta: Spionidae) from the Tay estuary. Metabolic response to severe hypoxia and hydrogen sulphide. Aquat. Ecol. 31,211 -222.
Sick, H. and Gersonde, K. (1969). Method of continuous registration of O2 binding curves of hemoproteins by means of a diffusion chamber. Anal. Biochem. 32,362 -376.[Medline]
Smith, E. B., Scott, K. M., Nix, E. R., Korte, C. and Fisher, C. R. (2000). Growth and condition of seep mussels (Bathymodiolus childressi) at a Gulf of Mexico Brine Pool. Ecology 81,2392 -2403.
Somero, G. N., Anderson, A. E. and Childress, J. J. (1989). Transport, metabolism and detoxification of hydrogen sulfide in animals from sulfiderich environments. Rev. Aquat. Sci. 1,591 -614.
Toulmond, A. (1992). Properties and functions of extracellular heme pigments. In Blood and Tissues Oxygen Carriers (ed. C. P. Mangum), pp.231 -256. Berlin: Springer Verlag.
Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. and
Jouin, C. (1990). Extracellular hemoglobins of hydrothermal
vent annelids: structural and functional characteristics in three alvinellid
species. Biol. Bull.
179,366
-373.
Tyuma, I., Kamigawara, Y. and Imai, K. (1973). pH dependence of the shape of the hemoglobinoxygen equilibrium curve. Biochim. Biophys. Acta 310,317 -320.[Medline]
Valentine, R. G., Shapiro, B. M. and Stadtman, E. R. (1968). Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli.Biochemistry 7,2143 -2152.[Medline]
Van Assendelft, O. W. (1970).Spectrophotometry of haemoglobin derivatives . Assen: Royal Vangorcum Ltd. 152pp.
Weber, R. E. (1972). On the variation in oxygen-binding properties of haemoglobins of lugworms (Arenicolidae, Polychaeta). In Proceedings of the Fifth European Marine Biology Symposium (ed. B. Battaglia), pp.231 -243. Padova, Italy: Editore Piccin.
Weber, R. E. (1978a). Respiratory pigments. In Physiology of Annelids (ed. P. J. Mill), pp.393 -446. London, New York, San Francisco: Academic Press.
Weber, R. E. (1978b). Respiration. In Physiology of Annelids (ed. P. J. Mill), pp.369 -392. London, New York, San Francisco: Academic Press.
Weber, R. E. (1980). Functions of invertebrate hemoglobins with special reference to adaptations to environmental hypoxia. Am. Zool. 20,79 -101.
Weber, R. E. (1981). Cationic control of O2 affinity in lugworm erythrocruorin. Nature 292,386 -387.
Weber, R. E., Malte, H., Braswell, E. H., Oliver, R. W. A., Green, B. N., Sharma, P. K., Kuchumov, A. and Vinogradov, S. N. (1995). Mass spectrometric composition, molecular mass and oxygen binding of Macrobdella decora hemoglobin and its tetramer and monomer subunits. J. Mol. Biol. 251,703 -720.[Medline]
Weber, R. E. and Vinogradov, S. N. (2001).
Nonvertebrate hemoglobins: Functions and molecular adaptations.
Physiol. Rev. 81,569
-628.
Wyman, J., Gill, S. J., Noll, L., Giardina, B., Colosimo, A. and Brunori, M. (1977). The balance sheet of a hemoglobin. Thermodynamics of CO binding by hemoglobin Trout I. J. Mol. Biol. 109,195 -205.[Medline]
Zal, F., Green, B. N., Lallier, F. H., Vinogradov, S. N. and Toulmond, A. (1997). Quaternary structure of the extracellular haemoglobin of the lugworm Arenicola marina. A multi-angle-laser-light-scattering and electrospray-ionisation-mass spectrometry analysis. Eur. J. Biochem. 243, 85-92.[Abstract]
Zal, F., Lallier, F. H., Wall, J. S., Vinogradov, S. N. and
Toulmond, A. (1996). The multi-hemoglobin system of the
hydrothermal vent tube worm Riftia pachyptila. I. Re-examination of
the number and masses of its constituents. J. Biol.
Chem. 271,8869
-8874.