Abstract
After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.
Highlights
Roles of distal and proximal heme pocket amino acids; (ii) the roles of internal water molecules near the active site; (iii) the roles of local and global conformational relaxations that are modulated by solvent; and (iv) the roles of pre-existing internal cavities associated with xenon binding
The reverse of these changes occurs after photolysis of bound carbon monoxide (CO), and extensive conformational relaxation is expected in the H64L/V68L double mutant
An expanded version of this “side path” scheme is shown in Fig. 11 and is based primarily on the kinetic consequences of mutating key amino acids located along proposed pathways, in the distal pocket, and adjacent to the Xe1 and Xe4 cavities [14]
Summary
Roles of distal and proximal heme pocket amino acids; (ii) the roles of internal water molecules near the active site; (iii) the roles of local and global conformational relaxations that are modulated by solvent; and (iv) the roles of pre-existing internal cavities associated with xenon binding. Over the past several years, we have developed the use of sol-gels and glycerol solutions that enhance geminate recombination by blocking ligand escape to the solvent These conditions provide a method to follow the evolution of the multiple kinetic phases for MbCO complexes and other hemeproteins by viscosity tuning at room temperatures in nonfrozen states [28, 30, 33]. After ligand dissociation in wild-type Mb, the imidazole side chain of His swings inward, occupies a position closer to the iron atom, inhibits internal rebinding, and eventually binds an internal water molecule in the equilibrium unliganded state, markedly inhibiting bimolecular rebinding from the solvent phase (Fig. 1, left panel). The E11 Ala, Val, Leu, and Phe series examines the effect of side chain size on ligand movement from the distal pocket to the remote xenon cavities and on access to the heme iron atom (Fig. 2). The side chain of Asn is less free to rotate because of the planar configuration of the amide group [50]
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