Structural characterization of the myoglobin active site using infrared crystallography

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Myoglobin binds and transports oxygen using a heme prosthetic group.
• Carbon monoxide (CO) is a known ligand and inhibitor of myoglobin.
• Accurate determination of ligand orientation within protein active sites is crucial for understanding binding mechanisms.

Purpose of the Study:

• To precisely determine the orientation of carbon monoxide (CO) bound to the myoglobin active site.
• To compare spectroscopic findings with existing structural models derived from X-ray and neutron diffraction.
• To elucidate the steric mechanisms that influence CO binding affinity to myoglobin.

Main Methods:

• Single crystals of myoglobin were used for measurements.
• Polarized infrared (IR) absorption spectroscopy was employed to probe CO orientation.
• The orientation of the C-O bond was determined relative to the heme plane.

Main Results:

• The C-O bond was found to lie approximately 7 degrees from the normal to the mean heme plane.
• This orientation contrasts significantly with larger angular displacements reported in diffraction-based models.
• The IR-derived orientation showed insensitivity to changes in pH or crystal packing.

Conclusions:

• The C-O bond orientation determined by IR spectroscopy suggests that diffraction-based structural models may be inaccurate.
• The small energy cost for a 7-degree displacement indicates protein distortion, not the Fe-C-O unit, is the primary steric barrier to CO binding.
• This study refines our understanding of ligand-protein interactions in myoglobin.