Respiratory adaptations in a deep-sea orbiniid polychaete from Gulf of Mexico brine pool NR-1: metabolic rates and hemoglobin structure/function relationships
Stéphane Hourdez1,2,*,
Roy E. Weber3,
Brian N. Green4,
John M. Kenney5 and
Charles R. Fisher1
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
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Fig. 1. Experimental apparatus used for respiration measurements at controlled
temperature.
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Fig. 2. Oxygen consumption for a specimen (1.24 g wet mass) of Methanoaricia
dendrobranchiata at 8 °C. (A) Changes in the oxygen concentration in
the chamber during the experiment. Readings were taken every 5 min. (B)
Calculated oxygen consumption rate as a function of oxygen concentration for
the same worm. Regression (i): r2=0.006, N=54,
P=0.589; regression (ii): r2=0.903,
N=20, P<0.0005. Pc, critical
pressure.
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Fig. 3. Relationship between oxygen consumption rate and wet mass of
Methanoaricia dendrobranchiata. The line fits the equation:
 O2=0.488M-0.702,
r2=0.457, P<0.1.
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Fig. 4. Percentage survival of Methanoaricia dendrobranchiata under
normoxia, anoxia, anoxia + 60 µmol l-1 sulfide and anoxia +1
mmol l-1 sulfide. Twenty-two worms were exposed to each
condition.
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Fig. 5. Hemoglobin content as a function of wet mass for Methanoaricia
dendrobranchiata (N=61). The dotted line fits the equation:
Q=0.256M0.056, P<0.01.
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Fig. 6. Evidence for the hexagonal bilayer (HBL) structure of Methanoaricia
dendrobranchiata hemoglobin. (A) Negatively stained electron microscope
image of M. dendrobranchiata hemoglobin. The dark areas are stain.
The protein of the molecular complex appears pale. The hemoglobin molecular
complexes are in various orientations. The top and side views (indicated)
appear to be similar to the top (sixfold) and side (twofold) views of other
annelid hemoglobins. Scale bar, 200 nm. (B) An image of a typical top view
taken from the electron microscope image in A. Scale bar, 30 nm. (C)
Rotational frequency analysis of image B showing the strong sixfold symmetry
expected of an HBL. (D) The sixfold rotationally Fourier-filtered image of B.
The sub-structure, such as the density in the middle and the holes within the
six subunits, is similar to that of other HBLs. Scale bar, 30 nm.
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Fig. 7. Example mass spectra (one of five) of the 3.5x106 Da
hemoglobin under (A) non-reducing, (B) reducing and (C) reduced and
carboxyamidomethylated (Cam) conditions. (D) Model of structure of the globin
monomers and dimers. The inset in A shows the linker dimers on the same
intensity scale as in A but on an expanded mass scale. a1-a4, b and c, globin
chain subunits; LD1-LD4, linker dimers; Dm, globin dimer; x2, portion of
the spectrum where intensity has been multiplied by 2.
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Fig. 8. Effect of pH on the affinity (P50 in mmHg; 1 mmHg=0.133 kPa) and
cooperativity (n50) of the intact hemoglobin (W-Hb) and putative
dodecameric fraction (D) at 10, 20 and 30°C. Numbers in italics show the
Bohr factors ( ) for the pH range 6.8-7.5. Buffer, 0.125 mol
l-1 Hepes in Riftia saline. Heme concentration, 0.51 mmol
l-1 (W-Hb) and 0.22 mmol l-1 (D).
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Fig. 9. Extended Hill plot of the intact hemoglobin at 20°C pH 6.77 and 7.60.
As indicated, the intersections between the lower and upper asymptotes to the
Hill plot with the y-axis at logPo2-0, indicate
the values of logKT and logKR, respectively. Other
conditions as in Fig. 8.
KR, oxygen-binding affinity of the hemoglobin in the oxygenated
(relaxed) state; KT, oxygen-binding affinity of the hemoglobin in
the deoxygenated (tense) state; Y, saturation.
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Fig. 10. Arrhenius plot for both the intact hemoglobin (W-Hb; open symbols) and the
dodecameric fraction (Dodec; filled symbols) at pH 6.8 and 7.6. Other
conditions as in Fig. 8.
P50, oxygen partial pressure at half-saturation; T, absolute
temperature. 1 mmHg=0.133 kPa.
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Fig. 11 . Example of the effect of [Ca2+] on the oxygen affinity
(P50 in mmHg) and cooperativity (n50) of the intact
hemoglobin and its dodecamers at pH 7.0. Heme concentration, 0.32 mmol
l-1. W-Hb, intact hemoglobin; D, dodecameric fraction. 1 mmHg=0.133
kPa. Open columns, [Ca2+]=0 mmol l-1; stippled columns,
[Ca2+]=0.1 mmol l-1.
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© The Company of Biologists Ltd 2002
