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Abstract
Myoglobin is a globular protein responsible for reversible binding and transport of
oxygen through the muscles of the body by use of an iron containing heme cofactor.
H
Co(II)Mb + O2
kon
koff
Co(III)Mb-O2
Vs.
Fe(II)Mb + O2
kon
koff
Fe(III)Mb-O2
The cobalt(II) analog of myoglobin can also reversibly bind molecular oxygen,
forming 1:1 adducts with this ligand. Studies have shown that oxygen binding
occurs at a comparable rate to that of the iron species, but there is a significant
difference between their rates of oxygen dissociation.
In this study, I explore the disparity in the rates of oxygen dissociation of the two
complexes in their conversion from the oxygenated to the deoxygenated forms.
There is expected to be a faster rate of dissociation for the cobalt analog due to
weaker binding of the oxygen to the metal center.
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Cobalt (II) Myoglobin Protein Structure
Protoporphyrin IX
heme
Histidine 64
Cobalt (II)
Histidine 93
CoIIMb
2
Active Sites of oxy-FeMb and oxy-CoMb
2.77 Å
2.95 Å
3.01 Å
2.72 Å
O2
2.06 Å
2.17 Å
Brucker, Eric A.; Olson, John S.; Phillips, George N. Jr. J. Bio. Chem. 1996, 271, 25419-25422
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Dissociation of Oxygen from Cobalt Myoglobin
chemed.chem.purdue.edu/.../1biochem/blood3.html
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Method
Na2S2O4
Oxymyoglobin was prepared by dissolving a measured amount in minimal
buffer, and adding excess sodium dithionite. It was then passed through a G25 Sephadex column for purification.
Known concentrations of both the hydrosulfite solution and the diluted
myoglobin species were mixed in a vial and immediately added to a cuvette,
where the reaction was monitored kinetically at predetermined wavelengths.
5
4
OxyCoMb
0.225
2
1.5
0.5
570
532
0.05
323
1
380
0.1
2.5
484
492
485
Absorbance (AU)
0.15
0.125
352
0
0.025
375
400
425
450
475
500
525
550
200
Wavelength (nm)
300
400
500
600
700
800
900
Wavelength (nm)
0.7
dd
2.5
571
0.5
556
Absorbance (AU)
3
538
0.6
3.5
0.4
0.3
0.2
0.1
2
0
520
1.5
N-band
350
560
580
600 Wavelength (nm)
571
0.5
0
538
556
482
492
485
1
540
Q-band
356
Absorbance (AU)
Absorbance (AU)
3
Porphyrin
лл*
0.175
0.075
DeoxyCoMb
3.5
0.2
555
0.25
406
426
Absorption Spectrum for oxyCoMb and deoxyCoMb
Soret-band
400
450
500
6
550
600
Wavelength (nm)
Crystal Field Splitting and Distortion
b1g (d x2-y2)
eg
a1g (d z2)
3d
eg (dxz,dyz)
t2g
Free metal
eg (dxy)
Tetragonal
field
Octahedral
field
d yz
d xz
Rhombic
field
A. Eaton and J. Hofrichter, in Methods in
Enzymology, Vol. 76, Academic Press, 1981.
7
Crystal Field Analysis
d x2-y2
d z2
d yz
d xz
d xy
d x2-y2
d x2-y2
d x2-y2
d z2
d z2
d z2
d yz
d yz
d yz
d xz
d xy
d xz
d xy
d xz
d xy
Deoxy-Fe(II)Mb
Oxy-Fe(II)Mb
Deoxy-Co(II)Mb
Oxy-Co(II)Mb
(3d6, s=2)
(3d6, s=0)
(3d7, s=1/2)
(3d7, s=1/2)
high spin
low spin
low spin
low spin
weak field
strong field
strong field
strong field
A. Eaton and J. Hofrichter, in Methods in
Enzymology, Vol. 76, Academic Press, 1981.
8
411
Absorption Spectrum for oxyMb  metMb
0.16
0.14
λmax OxyMb
Absorbance (AU)
0.12
0.1
543
λmax metMb
0.08
0.06
0
350
375
400
425
450
475
500
525
550
596
0.02
572
462
463
485
0.04
Wavelength (nm)
At low concentrations of dithionite (< 3.6 mM in solution), oxymyoglobin is
observed to convert to the metmyoglobin species, with release of superoxide,
rather than oxygen.
9
Absorption Spectrum of oxyMbdeoxyMb
433
0.16
0.14
Absorbance (AU)
0.12
0.1
0.08
0.06
0.02
0
400
425
450
475
578
584
591
596
488
485
0.04
500
525
550
575
Wavelength (nm)
At a high concentration of dithionite (≈ 12 mM in solution), oxymyoglobin is
observed to convert to the deoxygenated form, which indicates release of
oxygen rather than superoxide.
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Absorbance Changes
oxyCoMbdeoxyCoMb
0.3
0.25
Absorbance (AU)
426 nm
0.2
407 nm
0.15
0.1
555 nm
532 nm 571 nm
0.05
Isosbestic
point
0
400
500
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Wavelength (nm)
Kinetic Results (Cobalt Myoglobin)
Absorbance (AU)
1.7516 μM oxyCoMb + 12 mM sodium Dithionite
0.18
426 nm
0.16
(oxyCoMb)
0.14
0.12
0.1
407 nm
0.08
(deoxyCoMb)
0.06
100
200
300
400
500
Time(s)
1.7516 μM oxyCoMb + 3.6 mM sodium Dithionite
0.2
0.18
Absorbance (AU)
0.16
0.14
0.12
0.1
0.08
0.06
0.04
100
200
300
400
500
Time(s)
Measurements performed using a UV-Visible Spectrophotometer (pH 7.0, 22°C).
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Kinetic Results (Native Myoglobin)
1.2577 μM oxyMb + 3.6 mM sodium Dithionite
0.18
417 nm
0.16
(oxyMb)
Absorbance (AU)
0.14
0.12
409 nm
0.1
(deoxyMb)
0.08
0.06
0.04
0.02
100
200
300
400
500
Time(s)
1.1913 μM oxyMb + 1.2 mM sodium Dithionite
0.14
Absorbance (AU)
0.12
0.1
0.08
0.06
0.04
0.02
100
200
300
400
500
600
700
800
Time(s)
Measurements performed using a UV-Visible Spectrophotometer (pH 7.0, 22°C).
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Calculation of x
x(ε426nm OxyCoMb) + y(ε426nm deoxyCoMb) = A1/Ci
x(ε407nm OxyCoMb) + y(ε407nm deoxyCoMb) = A2/Ci
x is a fractional concentration and y= 1-x
 x(ε426nm OxyCoMb) + (1-x)(ε426nm deoxyCoMb) = A1/Ci
 x(ε426nm OxyCoMb) + (-x)(ε426nm deoxyCoMb) + (ε426nm deoxyCoMb)= A1/Ci
 x(ε426nm OxyCoMb- ε426nm deoxyCoMb) + (ε426nm deoxyCoMb)= A1/Ci
 x(ε426nm OxyCoMb- ε426nm deoxyCoMb) = A1/Ci - (ε426nm deoxyCoMb)
x=
A1/Ci - (ε426nm deoxyCoMb)
(ε426nm OxyCoMb- ε426nm deoxyCoMb)
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Approximation of Dissociation Rate Constant
(1.7516 μM) OxyCoMb  DeoxyCoMb
(1.2356 μM) OxyMb  DeoxyMb
-13.5
-14.1
2
2
y = -14.222 - 0.0010685x R = 0.99353
-14.2
y = -13.721 - 0.0115x R = 0.9943
Koff =
-14
-14.3
Koff =
(1.069 + 0.007) x 10-3 s-
s-
-14.4
ln (x)
(1.115 + 0.001) x
-14.5
10-2
ln(x)
t1/2 = 62 s
-15
t1/2 = 648 s
-14.5
-14.6
-14.7
-15.5
-14.8
-16
-14.9
0
50
100
150
200
Time (s)
0
100
200
300
400
500
Time (s)
Measurements were conducted using a UV-visible spectrophotometer
(22 °C, pH 7.0, 12 mM Sodium Dithionite)
At atmospheric levels of O2 (≈ 234 μM), the dissociation rate of the axial ligand at
the sixth coordinate position is approximately one order of magnitude faster in the
Cobalt containing analog compared to the native species.
15
600
Interaction between the Metal Center and Oxygen
His 64
His 64
His 64
O
Superoxide ion
O
CoIII
O
O
Na2S2O4, 2H+
CoII
o
pH 7.0, 22 C
His 93
His 93
CoII
+ O + H O + NaHSO 23
2
2 2
His 93
•Both the Cobalt and Iron metal centers have resonance forms which involve a
superoxide ion.
•Upon addition of the dithionite, numerous reactions may occur which include
release of oxygen, reduction of the metal, release of superoxide and its reaction
with two hydrogen ions to form hydrogen peroxide.
16
Possible Reaction of Fe in solution
FeII + HSO3O
SO2-
O
O
FeII
O
O2-
FeIII
+
2H+
III
Fe
O
O
FeIV
FeIV
Compound 1
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FeIII + H2O2
Compound 2
Conclusions
The studies of the dissociation of oxygen from the myoglobin analogs utilizing
sodium dithionite were unsuccessful for several reasons. The concentration of
dithionite was not great enough for the reaction to be pseudo first order. The
reaction occurs too fast at such concentrations. The lengthy reduction of the metal
species by dithionite and the use of an open system lead to the production of
numerous radicals and species in various oxidation states, resulting in complex
kinetic behavior.
The rate of dissociation of oxygen from the cobalt analog should have been on the
order of 103 s- while that of the native species should have been about two orders
of magnitude less, based on previous temperature jump relaxation analysis.
The dissociation of superoxide prior to reduction of the metal species by
hydrosulfite was observed, but only an approximate rate of dissociation could be
determined due to the complex nature of the reaction.
This experiment could be improved by using the stopped-flow apparatus at low
temperatures. Also, in place of hydrosulfite, a ligand which binds more strongly to
the myoglobin may be more appropriate in determination of the rate of oxygen
dissociation.
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References
[1] Hoffman, B. M.; Petering, D. H. Proc. Nat. Acad. Sci. 1970, 67, 637.
[2] Spilburg, Curtis A.; Hoffman, Brian M.; Petering, Davind H. J. Bio. Chem. 1972, 247, 42194223.
[3] Brucker, Eric A.; Olson, John S.; Phillips, George N. Jr. J. Bio. Chem. 1996, 271, 25419-25422.
[4] Matsuo, Takashi; Tsuruta, Takashi; Maehara, Keiko; Sato, Hideaki; Hisaeda, Yoshio; Hayashi,
Takashi. Inorg. Chem. 2005, 44, 9391-9396.
[5] Ikedai-Saito, Masao; Yamamoto, Haruhiko; Imai, Kiyohiro, Kayne, Frederick J.; Yonetani,
Takashi. J. Bio. Chem. 1977, 252, 620-624.
[6] Yonetani, Takashi. J. Bio. Chem. 1967, 242, 5008-5013.
[7] Charles Dickinson
[8] Alan Bruha
[9] (1)Yamamoto, Haruhiko; Kayne, Frederick J.; Yonetani, Takashi. J. Bio. Chem. 1974, 249, 691698.
(2) Yonetani, Takashi; Yamamoto, Haruhiko; Woodrow III, George V. J. Bio. Chem. 1974, 249,
682-690.
[10] Hambright, Peter, Lemelle, Stephanie. Inorganica Chimica Act, 92 (1984), 167-172.
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Rate Analysis of Oxygen Dissociation
from Native and Oxy-Cobalt Myoglobin
Advanced Inorganic Chemistry, Johns Hopkins University
3003 North Charles Street, Baltimore, MD 21218
[email protected]
Jamal N. Shillingford
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