LIGAND-BINDING TO HEME-PROTEINS .2. TRANSITIONS IN THE HEME POCKET OFMYOGLOBIN

Phenomena occurring in the heme pocket after photolysis of carbonmonox ymyoglobin (MbCO) below about 100 K are investigated using temperature -derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experime nts on MbCO solution samples at different pH and on orthorhombic cryst als. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A sub state. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilib rium populations of the B substates after photolysis below 25 K and de termine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those betw een A substates have only been observed above about 160 K. The transit ions between the B substates are nonexponential in time, providing evi dence for a distribution of substates. The temperature dependence of t he B substate transitions implies that they occur mainly by quantum-me chanical tunneling below 10 K. Taken together, the observations sugges t that the transitions between the B substates within the same A subst ate reflect motions of the CO in the heme pocket and not conformationa l changes. Geminate rebinding of CO to Mb, monitored in the Soret band , depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a popula tion shift among the substates and not from a change of the rebinding to an individual A substate.