“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.”

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.

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

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 protein ever to be seen at atomic 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 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.

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.

Oxygen molecule

Carbon monoxide molecule

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.

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. 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.

Lines showing the electric field around myoglobin

"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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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.

Heme Group: CPK Alpha helices: Orange

Backbone: Honeydew

3D Physical Model of Myoglobin Distal Histidine: CPK

Carbon Monoxide Ligand: CPK

Myoglobin molecule with heme group

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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.

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