Rate Analysis of Oxygen Dissociation from Native and Oxy-Cobalt Myoglobin
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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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Abstract

Rate Analysis of Oxygen Dissociation from Native and Oxy-Cobalt Myoglobin Advanced Inorganic Chemistry, Johns Hopkins University3003 North Charles Street, Baltimore, MD 21218 [email protected] N. Shillingford


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.

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.

H

Abstract

kon

Co(II)Mb + O2

Co(III)Mb-O2

koff

Vs.

kon

Fe(II)Mb + O2

Fe(III)Mb-O2

koff


Cobalt ii myoglobin protein structure

Cobalt (II) Myoglobin Protein Structure

Protoporphyrin IX

heme

Histidine 64

Cobalt (II)

Histidine 93

CoIIMb


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

Active Sites of oxy-FeMb and oxy-CoMb

Brucker, Eric A.; Olson, John S.; Phillips, George N. Jr. J. Bio. Chem. 1996, 271, 25419-25422


Dissociation of oxygen from cobalt myoglobin

Dissociation of Oxygen from Cobalt Myoglobin

chemed.chem.purdue.edu/.../1biochem/blood3.html


Method

Method

Na2S2O4

Oxymyoglobin was prepared by dissolving a measured amount in minimal buffer, and adding excess sodium dithionite. It was then passed through a G-25 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.


Absorption spectrum for oxycomb and deoxycomb

Absorption Spectrum for oxyCoMb and deoxyCoMb

OxyCoMb

DeoxyCoMb

Porphyrin лл*

d  d

Q-band

N-band

Soret-band


Crystal field splitting and distortion

b1g (d x2-y2)

eg

d yz

a1g (d z2)

d xz

3d

eg (dxz,dyz)

t2g

eg (dxy)

Free metal

Octahedral field

Tetragonal field

Rhombic field

Crystal Field Splitting and Distortion

A. Eaton and J. Hofrichter, in Methods in Enzymology, Vol. 76, Academic Press, 1981.


Crystal field analysis

Crystal Field Analysis

d x2-y2

d x2-y2

d x2-y2

d x2-y2

d z2

d z2

d z2

d z2

d yz

d yz

d yz

d yz

d xz

d xy

d xz

d xz

d xz

d xy

d xy

d xy

Deoxy-Fe(II)Mb

(3d6, s=2)

high spin

weak field

Oxy-Fe(II)Mb

(3d6, s=0)

low spin

strong field

Deoxy-Co(II)Mb

(3d7, s=1/2)

low spin

strong field

Oxy-Co(II)Mb

(3d7, s=1/2)

low spin

strong field

A. Eaton and J. Hofrichter, in Methods in Enzymology, Vol. 76, Academic Press, 1981.


Absorption spectrum for oxymb metmb

Absorption Spectrum for oxyMb  metMb

λmax OxyMb

543

λmax metMb

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.


Absorption spectrum of oxymb deoxymb

Absorption Spectrum of oxyMbdeoxyMb

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.


Absorbance changes oxycomb deoxycomb

Absorbance ChangesoxyCoMbdeoxyCoMb

426 nm

407 nm

555 nm

532 nm

571 nm

Isosbestic point


Kinetic results cobalt myoglobin

Kinetic Results (Cobalt Myoglobin)

1.7516 μM oxyCoMb + 12 mM sodium Dithionite

426 nm

(oxyCoMb)

407 nm

(deoxyCoMb)

1.7516 μM oxyCoMb + 3.6 mM sodium Dithionite

Measurements performed using a UV-Visible Spectrophotometer (pH 7.0, 22°C).


Kinetic results native myoglobin

Kinetic Results (Native Myoglobin)

1.2577 μM oxyMb + 3.6 mM sodium Dithionite

417 nm

(oxyMb)

409 nm

(deoxyMb)

1.1913 μM oxyMb + 1.2 mM sodium Dithionite

Measurements performed using a UV-Visible Spectrophotometer (pH 7.0, 22°C).


Calculation of x

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)


Approximation of dissociation rate constant

Approximation of Dissociation Rate Constant

(1.7516 μM) OxyCoMb  DeoxyCoMb

(1.2356 μM) OxyMb  DeoxyMb

Koff =

(1.069 + 0.007) x 10-3 s-

Koff =

(1.115 + 0.001) x 10-2 s-

t1/2 = 648 s

t1/2 = 62 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.


Interaction between the metal center and oxygen

Interaction between the Metal Center and Oxygen

Superoxide ion

  • 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.


Possible reaction of fe in solution

Possible Reaction of Fe in solution

Compound 1

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.

Conclusions


References

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, 4219-4223.

[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, 691-698.

(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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