Jakub Kostal &Steve SontumThesis Presentation ‘06

Courtesy ofwww.mcsrr.org

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O2/CO

Binding of CO/O2 to Fe in Heme

FeN

N

NN

CO ligand

Process of Ligand binding in Heme

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www.chemistry.wustl.edu

• Myoglobin’s ability to bind oxygen is readily poisoned by its stronger affinity for carbon monoxide

• The affinity for CO is greatly reduced compared to free heme

How does ligand surroundings in myoglobin’sHeme pocket influence ligand binding?

1. Sterics

Bound vs. free Heme:The Ultimate Puzzle

2. Electrostatic interactions

Heme Pocket for Dummies

Studying Electrostatic Effects: Vibration of CO bond

• Triple bond character causes high vibration stretching frequencies

(CO) used to characterize different conformers of the bound state

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•Equilibrium IR absorbance spectrum of bound CO shows the major sub states A0, A1 and A 3 are associated with CO stretching bands at 1966, 1945, and 1927cm-1

•Dispersion of A sub states thought to be caused by electrostatic interactions between the CO dipole and the imidazole chain of the distal His64, which assumes different dynamic conformations

Studying Electrostatic Effects: Vibration of CO bond

• Triple bond character causes high vibration stretching frequencies

(CO) used to characterize different conformers of the bound state•Equilibrium IR absorbance

spectrum of bound CO shows the major sub states A0, A1 and A 3 are associated with CO stretching bands at 1966, 1945, and 1927cm-1

•Dispersion of A sub states thought to be caused by electrostatic interactions between the CO dipole and the imidazole chain of the distal His64, which assumes different dynamic conformations

Models for electrostatic interactions in the CO complexes - amino acid

mutations

A (A3): Asn68 Mb (CO = 1938cm-1 ; Fe-C = 527cm-1) B (A1, A2): Wild-Type Mb (CO = 1945cm-1 ; Fe-C = 507cm-1)C (A0): Val64/Thr68 Mb (CO = 1984cm-1 ; Fe-C = 477cm-1)

Preliminary studies on our project:

Building a simple Theoretical Model

• Generation of a vibrational force field using RESP method for various heme analogs with bound CO ligand

• Classical MD model built• Dynamic Simulation of an out-of-

plane electric field using Li ions to predict changes in CO vibrations based on experimental observations

• Hypothesis:

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Li (+/-)

Li (-/+)

(CO) < (CO) Molecular Dynamics

Trajectory

Observed Vibrational Shifts

• Expected trends somewhat preserved only at high e. field intensities

1942

1944

1946

1948

1950

1952

1954

1956

1958

-100 -50 0 50 100 150

Charge (au)

Vib

. fr

eque

ncy (

1/c

m)

“Torquing” motion of the CO ligand

• Fe-C-O bond locked in one “torquing” mode throughout the dynamic trajectory at higher E. field intensities

• Dominant mode at higher E. field

• Torquing motion accounts for additional centripetal stretching of the CO bond

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Analysis of the torquing mode

• Fe-O angle to the normal () is greater than Fe-C angle to the normal of the heme plane (). This differences increases with reversed e. field

• Direction of the electric field changes and

• As the intensity of e. field increases, and increase as well

• As the temperature increases, both angles increase

Normal direction Reversed direction

X-axis: time (0.01ps) Y-axis: angle (degree)

MD Trajectory of Full Myoglobin

N

N

0

10

20

30

40

50

60

70

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100

A B

% o

f M

D t

raje

cto

ry t

ime

Out of heme pocket

Inside heme pocket

Conclusions and Future Work

• We have successfully generated RESP force field for CO heme model to study the effect of electrostatic fields on the vibration of CO.

• We have observed a toquing motion of the CO ligand induced by electrostatic fields of high intensities.

• We have analyzed 2ns MD trajectory of full myoglobin and observed that distal His64 spends 88% of the time inside and 12% outside of the heme pocket.

• Generate force fields for similar O-O and NO bound heme models (in progress)

Acknowledgments

SteveMeghan

Judy

References

•Spiro T. G., Kozlowski P. M. 1998. Discordant results on FeCO deformability in heme proteins reconciled by density functional theory. J. Am. Chem. Soc. 120: 4524-4525•Phillips G. N., Teodoro M. L., Tiansheng L., Smith B., Olson J. S.. 1999. Bound CO Is a Molecular Probe of Electrostatic Potential in the Distal Pocket of Myoglobin. J. Phys. Chem. B. 103: 8817-8829•Nienhaus K., Pengchi D., Kriegl J. M., Nienhaus G. U. 2003. Structural Dynamics of Myoglobin: Effect of Internal Cavities on Ligand Migration and Binding. Biochemistry. 42: 9647-9658

•Ray, G. B., X.-Y. Li, J. A. Ibers, J. L. Sessler, and T. G. Spiro. 1994. How far proteins bend the FeCO unit? Distal polar and steric effects in heme proteins and models. J. Am. Chem. Soc. 116: 162-176. •Rovira, C., K. Kunc, J. Hutter, P. Ballone, and M. Parrinello. 1997. Equilibrium geometries and electronic structure of iron-porphyrin complexes: a density functional study. J. Phys. Chem. A. 101:8914-8925.

Solvated WT Trajectory