Timothy A. Jackson

Mid-infrared vibrational spectrum of CO after photodissociation from heme: Evidence for a ligand docking site in the heme pocket of hemoglobin and myoglobin

Time‐resolved mid‐ir absorption spectra of CO at 283 K have been measured 100 ps after photodissociation from human hemoglobin A, horse myoglobin, and sperm whale myoglobin. The spectra reveal two vibrational features that are narrower than any reported for CO in the condensed phase near room temperature, indicating that CO becomes localized in a rotationally constrained environment. The integrated absorbance under these narrow features is 0.53±0.05 times that found for sperm whale myoglobin in low temperature glasses. A model is developed that relates this reduction of integrated absorbance to molecular motion in a rotationally constrained environment. From this model, the barrier to CO rotation is found to be 1.5±0.25 kcal/mol. The two vibrational features are tentatively assigned to CO oriented oppositely in the same site within the heme pocket. Evidently, the residues circumscribing the heme pocket in hemoglobin and myoglobin fashion a cavity near the binding site that accommodates the dissociated CO and restricts its rotational motion. This ‘‘docking’’ site mediates ligand transport to and from the active binding site and may be important to the function of ligand‐binding heme proteins.

Femtosecond IR spectroscopy: methods and applications to protein dynamics

We have developed a fs IR absorption spectrometer that provides high sensitivity in both near- and mid-IR regions. High sensitivity is attained using a dual beam approach: a fs broadband IR probe pulse is split into sample and reference pulses which are routed through and around the sample, respectively. The two pulses are directed through a monochromator before impinging on photodetectors.

Dynamics of conformational relaxation in myoglobin

The picosecond evolution of the tertiary conformation of myoglobin following photodissociation of carbonmonoxymyoglobin was investigated at room temperature by probing band III, a weak iron-porphyrin charge-transfer transition near 13110 cm-1 (763 nm) whose position is sensitive to the out-of-plane displacement of the iron. Upon photolysis, the iron moves out of the plane of the porphyrin causing a blue shift of band III and a concomitant change in the protein conformation. The conformational relaxation reveals a viscosity dependence even at early times (< 2 ps), indicating that the primary motion of the protein involves a displacement of the surrounding solvent. This motion likely corresponds to a displacement of the F-helix. The ensuing relaxation is highly nonexponential, in agreement with recent molecular dynamics simulations. The conformational changes occurring near the heme likely affect the height of the barrier to ligand rebinding and may explain nonexponential rebinding of ligands at ambient temperatures.