After photodissociation of carbon monoxide bound to myoglobin, the protein relaxes to the deoxy equilibrium structure in a quake-like motion. Investigation of the proteinquake and of related intramolecular equilibrium motions shows that states and motions have a hierarchical glasslike structure.The dynamic aspects of proteins have been studied extensively in recent years and a picture of ever increasing complexity has emerged. To bring some order into the complexity, we have introduced a model that classifies states and motions (1). In the present paper, we describe the model and its experimental basis in more detail. STATES, SUBSTATES, AND MOTIONSWe consider myoglobin (Mb), an oxygen storage protein, consisting of 153 amino acids, with molecular weight of 17,900 and approximate dimensions of2.5 x 4.4 x 4.4 nm (2). Embedded in the protein matrix is a heme group with a central iron atom, which binds small ligands such as dioxygen (02) or carbon monoxide (CO) reversibly. Thus, two states are involved in the function of Mb, deoxyMb and liganded Mb (e.g., MbCO). In the liganded state, the heme is planar and the iron has spin 0 and lies close to the mean heme plane. In the unliganded state, the heme group is domed, the iron has spin 2 and lies =0.5 A away from the mean heme plane, and the globin structure differs somewhat from the liganded one (3).A protein molecule in a particular state can assume a very large number of conformational substates (CS) (4-6). Different substates have the same overall structure, but they differ in details; they perform the same function, but with different rates. equilibrium (10). Return to equilibrium occurs through a proteinquake: the released strain energy is dissipated through waves [phonons (11) or solitons (12)1 and through the propagation of a deformation (2, 3). HIERARCHY OF SUBSTATESThe experiments described in the next section imply that the proteinquake released by photodissociation of MbCO propagates sequentially: Fig. 2, consequently is much more complex than we originally anticipated (4).The valley in the top diagram of Fig. 2a represents one state, say MbCO. MbCO can exist in a large number of conformational substates, CS1, separated by high barriers.Each valley in the first tier is structured into substates (CS2) with smaller barriers. The furcation continues through two more tiers, with decreasing barrier heights. The dynamic

We have studied the infrared spectra of the bound and photodissociated states ofMb-"2CO and Mb-'3CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-'2CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-'. The low-temperature photodissociated state (Mb*) shows three lines (BO, B1, B2) at 2144, 2131, and 2119 cm-' for "2CO. The absorbances of the three lines depend on temperature. Bo is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb* have near-infrared, EPR, and Mossbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb* and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.The reversible binding of CO to the storage protein Mb can be studied with flash photolysis (1). Experiments in which the Soret line was monitored demonstrate that the binding process involves a number ofsteps (2, 3). Here we show that monitoring the CO stretching vibration reveals additional features of the protein's interior.The active center of Mb, the heme group, is embedded in the protein (Fig. 1) (4) and the ligand binds at the central heme iron. In flash photolysis, the bound-state MbCO is photodissociated. Below 200 K the CO cannot leave the heme pocket and rebinds from there. Two states are involved in low-temperature recombination: state A, in which the CO is bound, the heme is nearly planar, and the iron atom has spin 0; and state B, in which the CO is photodissociated from the heme iron and remains in the protein pocket and the iron has spin 2. At low temperatures, the rebinding process B to A is not exponential in time. We have explained this observation by postulating the existence of conformational substates (2, 5). At low temperatures each Mb molecule is frozen into a particular substate with a specific barrier height for rebinding. From 180 to 80 K the transition occurs by an over-the-barrier Arrhenius process; below 60 K, quantum mechanical tunneling dominates (6, 7). State B (Mb*) has been studied in MbCO and CoMbCO by near-infrared (8, 9), EPR (10, 11), and Mbssbauer (12) Pentex (Kankakee, IL) was dissolved in 70% (vol/vol) glycerol in water buffered to pH 7 with 0.1 M phosphate. The sample was stirred under a CO atmosphere for several hours, reduced with sodium dithionite, and stirred for s...

The recombination after flash photolysis of dioxygen and carbon monoxide with sperm whale myoglobin (Mb), and separated beta chains of human hemoglobin (beta A) and hemoglobin Zürich (beta ZH), has been studied as a function of pH and temperature from 300 to 60 K. At physiological temperatures, a preequilibrium is established between the ligand molecules in the solvent and in the heme pocket. The ligand in the pocket binds to the heme iron by overcoming a barrier at the heme. The association rate is controlled by this final binding step. The association rate of CO to Mb and beta A is modulated by a single titratable group with a pK at 300 K of 5.7. The binding of CO to beta ZH, in which the distal histidine is replaced by arginine, does not depend on pH. Oxygen recombination is independent of pH in all three proteins. Comparison of the binding of CO at 300 K and at low temperatures shows that pH does not affect the preequilibrium but changes the barrier height at the heme. The pH dependence and the difference between O2 and CO binding can be explained by a charge-dipole interaction between the distal histidine and CO.

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