Motions in Myoglobin

When the structure of myoglobin was first revealed by x-ray crystallography, it was discovered that there was no clear evidence of a pathway for ligands to enter the protein to bind at the heme iron. Motions within the protein are necessary for the protein to function. Pressure release and flash photolysis experiments help characterize some of these protein motions. Sperm whale carbonmonoxymyoglobin exhibits three spectroscopically distinguishable carbon monoxide stretch bands between 1910 cm^{-1} and 1990 cm^{-1} labeled A_0, A_1, and A_3. Pressure release measurements reveal three relaxations in the protein which are measured by: (1) the shifting in frequency of A_0 , and the exchanges (2) A_1 to A_3, and (3) A_{0} to A_{1} + A_{3} . Each relaxation is non-Arrhenius and solvent viscosity dependent. The rebinding of the A substates was observed after flash photolysis by monitoring the Soret band and the A substates. The low-temperature rebinding (20K-160K) exhibit increasing rebinding rates with increasing temperature. The three A substates show different rebinding rates. From ~170K to ~220K, the rebinding rate decreases with increasing temperature in each A substate. Between 220K and ~ 300K, the absorbance change of A_0 is non-monotonic in time due to an interconversion between A_0 and A_ {1} + A_3 during rebinding. This interconversion rate matches well with the extrapolated interconversion rate deduced from pressure release measurements. Since the three A substates have different rebinding rates, it is possible that the binding process is controlled in part by the interconversion between A substates. The maximum entropy method applied to the rebinding data reveals five peaks in the distribution of rebinding rates, labeled (from fast to slow) 1,2,3,E, and S. Peak 1 is due to geminate rebinding. Peaks 2 and 3 are well characterized, but not well understood. Peak 2 is present in all the A substates (except possibly A_3 ) with the same rate as peak 2 monitored in the Soret. Peak 3 is the same in the Soret, A_1 and likely A_3, however, it is absent in A_0. Peak 3 matches closely with the extrapolated interconversion between A_1 and A_3 and the shifting of peak frequency of A_0 measured in pressure release experiments. Peak E is due to the interconversion A_{0 } to A_{1} + A_{3 } and peak S arises from the rebinding of CO from the solvent.