Myoglobin is a protein used in animal muscle to store oxygen. (It is a close relative of hemoglobin, which carries oxygen in the blood.) The oxygen in both hemoglobin and myoglobin is bound to a large nonprotein molecule called a heme, which is tightly bound to the protein itself.

The heme binding site consists of a complex ring structure (a porphyrin) which surrounds an iron atom that is able to form six bonds. Four of the six iron bonding sites are occupied by nitrogens of the porphyrin ring, forming a planar structure (see above figure), one site is used to attach the heme group to the protein backbone, and the final site binds molecular oxygen. The heme group can bind other molecules besides oxygen; for example, carbon monoxide (CO) binds to heme–in fact, more strongly than does O₂.

The binding of CO is of great interest because carbon monoxide poisoning claims the lives of many people each year. Because the binding of CO to heme is so strong, even a small amount of CO in the atmosphere quickly saturates the heme binding sites. When this occurs, O₂ cannot bind to hemoglobin, the brain becomes starved for oxygen, and death occurs. Hence the interest in the processes by which CO associates with and dissociates from the heme unit.

A recent report in Chemical and Engineering News (1) highlights experiments on CO-heme association by Philip A. Anfinrud and coworkers (2) at Harvard University. These workers used femtosecond infrared spectroscopy, that is, infrared spectroscopy on an ultrafast time scale, to gain insight into the dissociation of CO from the heme group. Infrared spectroscopy measures the pattern of energy absorption when a substance is exposed to different wavelengths of infrared light. The energy absorbed is related to various vibrations excited in the molecule observed, and these vibrations can be correlated with various structural features of the molecules being studied. A femtosecond is one quadrillionth of a second (10^-15 seconds), a very short period of time, short enough that it is on the same scale as the rate of dissociation of the CO-heme complex. The time-resolved IR spectroscopic method involves taking absorbance measurements every few femtoseconds so that even the fastest reaction can be observed.

Using femtosecond infrared spectroscopy, the Harvard group found that CO dissociates from the heme by rotating approximately 90 degrees and falling into one of two orientations in a docking site near the hydrophobic center. The CO can detach and move to the docking site either with its carbon end facing towards the heme center or away from it (as in the example shown below).

This process occurs in less than one picosecond (10^-12 seconds, one trillionth of a second.) In the orientation shown above, the CO is hindered from returning to the heme site and it can then begin the slower process of being released by the myoglobin. The hindrance occurs because the protein actually changes its local structure so that any return of the CO to the heme binding site is blocked. Reactions of this speed are too fast to be observed even by new techniques such as nanosecond X-ray technology (3), and femtosecond infrared (IR) spectroscopy is so far the only method that gives us the opportunity to look at events involving protein structure and function on a such a rapid time scale. IR is not routinely used in this way to study proteins or their reactions; however, the strong IR absorption of CO makes this system an excellent one to study via femtosecond spectroscopy.

The researchers feel that this technique can give good insight into processes of dissociation that can occur in other proteins whose activity involves binding of oriented molecules both in and out of the active site. Myoglobin is a well-studied protein and is an example of what most likely happens in more complex systems. Since myoglobin is such a well-studied and understood structural system, success in this case means that the technique can be extended to less understood ligand-protein systems or more complex systems (e.g. hemoglobin) in order to characterize them as well.

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