Comparative Rate Analysis of Oxygen Dissociation in Native vs. Oxy-Cobalt Myoglobin

1. Rate Analysis of Oxygen Dissociation from Native and Oxy-Cobalt Myoglobin Advanced Inorganic Chemistry, Johns Hopkins University3003 North Charles Street, Baltimore, MD 21218 jshilli3@jhem.jhu.eduJamal 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. H Abstract kon Co(II)Mb + O2 Co(III)Mb-O2 koff Vs. kon Fe(II)Mb + O2 Fe(III)Mb-O2 koff

3. Cobalt (II) Myoglobin Protein Structure Protoporphyrin IX heme Histidine 64 Cobalt (II) Histidine 93 CoIIMb

4. 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

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 OxyCoMb DeoxyCoMb Porphyrin лл* d  d Q-band N-band Soret-band

8. 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.

9. 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.

10. 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.

11. 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.

12. Absorbance ChangesoxyCoMbdeoxyCoMb 426 nm 407 nm 555 nm 532 nm 571 nm Isosbestic point

13. 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).

14. 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).

15. 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. 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.

17. 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.

18. Possible Reaction of Fe in solution Compound 1 Compound 2

19. 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

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