1. 1 Rate Analysis of Oxygen Dissociation from Native and Oxy-Cobalt Myoglobin Advanced Inorganic Chemistry, Johns Hopkins University 3003 North Charles Street, Baltimore, MD 21218 jshilli3@jhem.jhu.edu Jamal N. Shillingford

2. 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-O 2 Co(II)Mb + O 2 k on k off Fe(III)Mb-O 2 Fe(II)Mb + O 2 k off k on Vs.

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

4. 4 Active Sites of oxy-FeMb and oxy-CoMb O2O2 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. 5 Dissociation of Oxygen from Cobalt Myoglobin chemed.chem.purdue.edu/.../1biochem/blood3.html

6. 6 Method Na 2 S 2 O 4 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. 7 Absorption Spectrum for oxyCoMb and deoxyCoMb OxyCoMbDeoxyCoMb Porphyrin л  л* N-bandSoret-band Q-band d  d

8. 8 Crystal Field Splitting and Distortion A. Eaton and J. Hofrichter, in Methods in Enzymology, Vol. 76, Academic Press, 1981. egeg t 2g b 1g (d x 2 -y 2 ) a 1g (d z 2 ) e g (d xz,d yz ) e g (d xy ) 3d Free metalOctahedral field Tetragonal field Rhombic field d yz d xz

9. 9 Crystal Field Analysis Deoxy-Fe(II)Mb (3d 6, s=2) high spin weak field d x 2 -y 2 d z 2 d yz d xz d xy d x 2 -y 2 d z 2 d yz d xz d xy Oxy-Fe(II)Mb (3d 6, s=0) low spin strong field d x 2 -y 2 d z 2 d yz d xz d xy Deoxy-Co(II)Mb (3d 7, s=1/2) low spin strong field d x 2 -y 2 d z 2 d yz d xz d xy Oxy-Co(II)Mb (3d 7, s=1/2) low spin strong field A. Eaton and J. Hofrichter, in Methods in Enzymology, Vol. 76, Academic Press, 1981.

10. 10 Absorption Spectrum for oxyMb  metMb 543 λ max OxyMb λ max 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. 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. 12 Absorbance Changes oxyCoMb  deoxyCoMb Isosbestic point 426 nm 407 nm 532 nm571 nm 555 nm

13. 13 Kinetic Results (Cobalt Myoglobin) 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 1.7516 μM oxyCoMb + 3.6 mM sodium Dithionite

14. 14 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

15. 15 Calculation of x x(ε 426nm OxyCoMb ) + y(ε 426nm deoxyCoMb ) = A 1 /C i x(ε 407nm OxyCoMb ) + y(ε 407nm deoxyCoMb ) = A 2 /C i x is a fractional concentration and y= 1-x  x(ε 426nm OxyCoMb ) + (1-x)(ε 426nm deoxyCoMb ) = A 1 /C i  x(ε 426nm OxyCoMb ) + (-x)(ε 426nm deoxyCoMb ) + (ε 426nm deoxyCoMb )= A 1 /C i  x(ε 426nm OxyCoMb - ε 426nm deoxyCoMb ) + (ε 426nm deoxyCoMb )= A 1 /C i  x(ε 426nm OxyCoMb - ε 426nm deoxyCoMb ) = A 1 /C i - (ε 426nm deoxyCoMb )  x = A 1 /C i - (ε 426nm deoxyCoMb ) (ε 426nm OxyCoMb - ε 426nm deoxyCoMb )

16. 16 Approximation of Dissociation Rate Constant At atmospheric levels of O 2 (≈ 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 K off = (1.115 + 0.001) x 10 -2 s - K off = (1.069 + 0.007) x 10 -3 s - (1.2356 μM) OxyMb  DeoxyMb t 1/2 = 62 s t 1/2 = 648 s

17. 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. 18 Possible Reaction of Fe in solution Compound 1Compound 2

19. 19 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 10 3 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.

20. 20 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.