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Myoglobin: O2 or Not O2… That is the Question
The Brown Deer SMART Team: Evan Bord, Robert Laughlin, Andrew LeMense, Chad Marable, Suzie Mielke, Brett Poniewaz, Virginia Tuncel, Gina Wade, Mike Weeden, Ryan Wisth
Teacher: David Sampe
Mentors: Hannah Wagie, Ph.D. Candidate and Peter Geissinger, Ph.D., Department of Chemistry and Biochemistry, University of Wisconsin - Milwaukee
Free divers can’t hold their breath as long as whales, but they train their
bodies to maximize their oxygen (O2) storing potential using the protein
myoglobin. Myoglobin’s structure has been known for decades, but
researchers are still trying to determine just how myoglobin functions.
Found in muscle tissue, myoglobin stores O2, a molecule needed to
produce chemical energy. Toxic ligands, such as carbon monoxide (CO)
and cyanide, also bind to myoglobin. When CO binds to a free heme
group, the heme's binding affinity for CO is 20,000 times that for O2.
When heme is surrounded by myoglobin, that binding affinity ratio drops
to only 25. The decrease was thought to be due to steric interactions
which prevented CO from occupying the same space as His64. Recent
evidence suggests that electrostatic interactions and hydrogen bonds play
a more important role. The O2 is stabilized as opposed to the CO being
pushed out. Several amino acids (His64, Val68, Phe43, Phe46, and
Leu29,) seem to stabilize the ligand. With 3D printing technology, the
Brown Deer SMART (Students Modeling a Research Topic) Team, funded
by a grant from NIH-CTSA, created a model of myoglobin. If researchers
can fully understand ligand discrimination by heme proteins, not only will
divers be able to hold their breath longer, but we may be able to remedy
conditions like hypoxia where there is a lack of O2 in the blood.
Scientists originally thought that the drastic difference in binding affinities between O2
and CO in a free heme compared to a heme protein on myoglobin was due to steric
interactions. Amino acid residues in the heme proteins push CO, bending it and
preventing it from sticking straight up. A protein residue in heme proteins close to the
ligand called the distal histidine forces CO into a bent position. O2, however, is not
affected by the distal histidine’s force because it already binds in a bent position. The
bending of CO prevents the ligand from occupying the same space as His64. The ratio of
CO to O2 binding affinities decreases when bound to myoglobin compared to when bound
to free heme.
3D Physical Model of
Myoglobin
Carbon Monoxide Ligand: CPK
Heme groups bind with several molecules
such as CO and O2. However, the heme
group’s affinity for CO is 20,000 times
greater than its binding affinity for O2. But,
when myoglobin is with the heme group, the
affinity for CO decreases to only 25 times
that of O2. Even though myoglobin still
favors CO, only 1 out of every 100,000
molecules in the atmosphere is CO.
Therefore, there is enough O2 in the
atmosphere to make up for oxygen’s lower
binding affinity. Current scientific research is
focused on determining why myoglobin
prefers O2 to CO.
Lines showing the electric
field around myoglobin
Scientists are studying the effect of myoglobin’s electric field on its active-site molecule, heme. Each amino
acid in the myoglobin chain contributes its own small effect on heme. This electric field will also affect the
interaction of myoglobin with potential ligands. In order to visualize the electric field, researchers use a
hole burning spectroscopy table.
Backbone: Honeydew
Alpha helices: Orange
16000
14000
12000
10000
CO:O2
Elephant seals, whales, and many
other aquatic mammals are able to
dive, in some cases, to even a mile
under water. They are able to achieve
these impressive feats because they
have a higher proportion of myoglobin
to hemoglobin than humans. Myoglobin
stores O2 in muscles, while hemoglobin
carries the oxygen from the lungs to the
rest of the body. Myoglobin was the first
Myoglobin
protein ever to be seen at atomic
molecule with
heme group
resolution by researchers, as early as the
1950’s. Because of myoglobin’s long
historical background in science, it has been quite thoroughly
studied. However, there is still a question about myoglobin not
yet laid to rest. Researchers are still trying to figure out why
myoglobin has such a high affinity for O2. This high affinity is
what keeps all vertebrates alive, and allows aquatic mammals
to dive deep in order to get food.
Heme Group: CPK
Distal Histidine: CPK
According to current data, steric hindrance is not the only factor in determining
ligand binding. Therefore, today’s research is focusing on electrostatic fields
surrounding myoglobin molecules. Every molecule consists of a collection of
electrical charges: positively charged nuclei and a negatively charged electron
cloud. Every electrical charge creates an electric field that can exert a force on
other charges. All the charges on a protein combine to form a electric field
around and within myoglobin. This electrostatic field determines how a ligand
binds to myoglobin. Electric fields are measured indirectly by measuring
absorption spectra from myoglobin when bombarded with different
wavelengths of light using hole burning spectroscopy.
8000
6000
4000
2000
0
Mutations of Mb from Sperm Whales.
In the wild type at position 64, there is a
distal histidine, but this can be exchanged
for different amino acids (as shown in
graph). This changes the ratio of
myoglobin’s binding affinity for CO to O2.
The amino acid mutants are organized
from small to large, illustrating that size
has minimal effect on the ratio of binding
affinity between O2 and CO, and in fact
has more to do with distance and charge
of the mutated residues.
Using hole burning spectroscopy, scientists measure the electric field indirectly by determining what
wavelengths of light are absorbed by Mb. In this Mb, heme is replaced with protoporphyrin IX, which is
identical to heme, but has no iron. This is necessary because heme does not fluoresce. Scientists use a
device called a pump laser to generate a beam, which a dye laser then converts to a wavelength of 620nm.
In order for light to be absorbed by protoporphyrin IX, the heme needs to be held still. This is achieved
using a cryostat, which brings the temperature inside to 1K, nearly stopping molecular movement. As the
beam passes through the sample, the absorption spectrum is measured with photomultiplier tubes (PMT).
The data produced by this procedure is processed by computers, eventually yielding values for the internal
electric fields.
Hole burning spectroscopy is a relatively new way to study proteins, but scientists are hopeful it could
lead to new breakthroughs in synthesizing blood substitutes, understanding industrial protein catalysts,
and understanding how electrons are transported in photosynthesis. Many of the studies conducted with
hole burning spectroscopy are not simply to find out more about myoglobin, but are also meant to test
out the technique. Someday, hole burning spectroscopy might be used on more proteins, and electric
fields may become a more important data set for each protein.
Carbon
monoxide
molecule
Oxygen
molecule
"The effect of a point mutation in the myoglobin active site on the electric field vector at the heme iron is
explored. In this model, only four active site residues are considered: PHE 43, HIS 64, VAL 68, HIS 93.
Each atom in these amino acids is replaced by a point charge. The coordinate of each point charge is from
the respective Protein Data Bank file (2MGK, 2MGA, 2MGC); the positive or negative partial atomic charge
of each point charge was given by the residue-specific value as published by Cornell, et al. (1995) using
the AMBER molecular dynamics package. Then, an energy job is run using the Gaussian09 computational
chemistry program that requests the electric field at the heme iron position (keyword: PROP=FIELD).
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2. S. Borman (1999). A Mechanism Essential to Life. Chemical & Engineering News 77: 31-364.
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http://themedicalbiochemistrypage.org/hemoglobin-myoglobin.php
4. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; … Fox, D. J. Gaussian 09,
Revision A.02, Gaussian, Inc.: Wallingford, CT, 2009.
5. Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Kenneth M. Merz, J.; … Kollman, P. A., A Second Generation Force
Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules. Journal of the American Chemical Society
1995, 117, 5179-5197.
“The SMART Team Program is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its
contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.”