1 Rate Analysis of Oxygen Dissociation from Native and Oxy-Cobalt Myoglobin Advanced Inorganic Chemistry, Johns Hopkins University 3003 North Charles Street,

1

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

2

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.

Abstract

H

Co(III)Mb-O2Co(II)Mb + O2

kon

koff

Fe(III)Mb-O2Fe(II)Mb + O2koff

kon

Vs.

3

CoIIMb

heme

Cobalt (II)

Histidine 93

Histidine 64

Protoporphyrin IX

Cobalt (II) Myoglobin Protein Structure

4

Active Sites of oxy-FeMb and oxy-CoMb

O2

2.17 Å

2.06 Å

2.77 Å

3.01 Å

2.95 Å

2.72 Å

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

5

Dissociation of Oxygen from Cobalt Myoglobin

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

6

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.

7

Absorption Spectrum for oxyCoMb and deoxyCoMb

Wavelength (nm)375 400 425 450 475 500 525 550

Abs

orba

nce

(AU

)

0.025

0.05

0.075

0.1

0.125

0.15

0.175

0.2

0.225

0.25

42

6

35

2

53

2

57

0

38

0

Wavelength (nm)200 300 400 500 600 700 800 900

Abs

orba

nce

(AU

)

0

0.5

1

1.5

2

2.5

3

3.5

4

40

6

55

5

48

54

92

48

432

3

Wavelength (nm)350 400 450 500 550 600

Abs

orba

nce

(AU

)

0

0.5

1

1.5

2

2.5

3

3.5

35

6

53

8

57

1

48

54

92

48

2 55

6

Wavelength (nm)520 540 560 580 600

Abso

rban

ce (A

U)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

538

571

556

OxyCoMb DeoxyCoMb

Porphyrin лл*

N-band Soret-band

Q-band

d d

8

Crystal Field Splitting and Distortion

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

eg

t2g

b1g (d x2-y

2)

a1g (d z2)

eg (dxz,dyz)

eg (dxy)

3d

Free metal Octahedral field

Tetragonal field

Rhombic field

d yz

d xz

9

Crystal Field Analysis

Deoxy-Fe(II)Mb

(3d6, s=2)

high spin

weak field

d x2-y

2

d z2

d yzd xz

d xy

d x2-y

2

d z2

d yz

d xz

d xy

Oxy-Fe(II)Mb

(3d6, s=0)

low spin

strong field

d x2-y

2

d z2

d yz

d xz

d xy

Deoxy-Co(II)Mb

(3d7, s=1/2)

low spin

strong field

d x2-y

2

d z2

d yz

d xz

d xy

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.

10

Absorption Spectrum for oxyMb metMb

543

Wavelength (nm)350 375 400 425 450 475 500 525 550

Abs

orba

nce

(AU

)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16 411

485

463

596

572

462

λmax OxyMb

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.

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Absorption Spectrum of oxyMbdeoxyMb

Wavelength (nm)400 425 450 475 500 525 550 575

Abs

orba

nce

(AU

)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

433

485

584

578

488

596

591

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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Wavelength (nm)400 500

Ab

sorb

an

ce (

AU

)

0

0.05

0.1

0.15

0.2

0.25

0.3

Absorbance ChangesoxyCoMbdeoxyCoMb

Isosbestic point

426 nm

407 nm

532 nm 571 nm

555 nm

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Kinetic Results (Cobalt Myoglobin)

Time(s)100 200 300 400 500

Abs

orba

nce

(AU

)

0.06

0.08

0.1

0.12

0.14

0.16

0.18

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

426 nm

(oxyCoMb)

407 nm

(deoxyCoMb)

1.7516 μM oxyCoMb + 12 mM sodium Dithionite

Time(s)100 200 300 400 500

Abs

orba

nce

(AU

)

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

1.7516 μM oxyCoMb + 3.6 mM sodium Dithionite

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Time(s)100 200 300 400 500

Abs

orba

nce

(AU

)

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

Kinetic Results (Native Myoglobin)

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

417 nm

(oxyMb)

409 nm

(deoxyMb)

1.2577 μM oxyMb + 3.6 mM sodium Dithionite

1.1913 μM oxyMb + 1.2 mM sodium Dithionite

Time(s)100 200 300 400 500 600 700 800

Abs

orba

nce

(AU

)

0.02

0.04

0.06

0.08

0.1

0.12

0.14

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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)

16

-16

-15.5

-15

-14.5

-14

-13.5

0 50 100 150 200

y = -13.721 - 0.0115x R2= 0.9943

ln(x

)

Time (s)

Approximation of Dissociation Rate Constant

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.

Measurements were conducted using a UV-visible spectrophotometer (22 °C, pH 7.0, 12 mM Sodium Dithionite)

(1.7516 μM) OxyCoMb DeoxyCoMb

Koff =

(1.115 + 0.001) x 10-2 s-

-14.9

-14.8

-14.7

-14.6

-14.5

-14.4

-14.3

-14.2

-14.1

0 100 200 300 400 500 600

y = -14.222 - 0.0010685x R2= 0.99353

ln (

x)Time (s)

Koff =

(1.069 + 0.007) x 10-3 s-

(1.2356 μM) OxyMb DeoxyMb

t1/2 = 62 s t1/2 = 648 s

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Interaction between the Metal Center and Oxygen

Superoxide ion

CoII

O

O

His64

His93

Na2S2O4, 2H+

CoIII

O

O

His64

His93

CoII

His64

His93

+ O2+ H2O2

+ NaHSO32-

pH 7.0, 22oC

•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.

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Possible Reaction of Fe in solution

Compound 1 Compound 2

FeIII

OO

FeII

OO

O2- + FeIII 2H+

FeIII+ H2O2

FeIV

O

FeIV

O

SO2-

FeII + HSO3-

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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, 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.