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We have recently reported spectroscopic evidence for structural relaxation of myoglobin (Mb) following photodissociation of MbCO [Lambright, D. G., Balasubramanian, S., & Boxer, S. G. (1991) Chem. Phys. 158, 249-260]. In this paper we report measurements for a series of single amino acid mutants of human myoglobin on the distal side of the heme pocket (positions 45, 64, and 68) in order to examine specific structural determinants involved in this conformational relaxation and to determine the nature of the coupling between relaxation and the functional process of ligand binding. The kinetics of ligand binding and conformational relaxation were monitored by transient absorption spectroscopy in the Soret spectral region, and the results are analyzed using a four-state ligand binding model. Two principal results emerge: (1) amino acid substitutions in the distal heme pocket affect the kinetics of the nonequilibrium conformational relaxation and (2) the rate of ligand escape from the protein matrix is not significantly perturbed by the distal heme pocket mutations.

The infrared spectra of CO bound to human myoglobin and myoglobin mutants at positions His-64, Val-68, Asp-60, and Lys-45 on the distal side have been measured between 100 and 300 K. Large differences are observed with mutations at His-64 and Val-68 as weli as with temperature and pH. Although distal His-64 is found to affect CO bonding, Val-68 also plays a major role. The variations are analyzed qualitatively in terms of a simple model involving steric interaction between the bound CO and the distal residues. A strong correlation is found between the final barrier height to CO recombination and the CO stretch frequency: as compared to wild type, the barrier is smaller in those mutants that have a higher CO stretch frequency (vCO) and vice versa. Possible reasons for this correlation are discussed. It is emphasized that the temperature and pH dependence of both the kinetics and the infrared spectra must be measured to obtain a consistent picture.

In order for diatomic ligands to enter and exit myoglobin, there must be substantial displacements of amino acid side chains from their positions in the static X-ray structure. One pathway, involving Arg/Lys45, His64, and Val68, has been studied in greatest detail. In an earlier study (Lambright et al., 1989) we reported the surprising result that mutation of the surface residue Lys45 to arginine lowers the inner barrier to CO rebinding. Until then, it had been thought that this barrier primarily involves interior distal pocket residues such as His64 and Val68. In this report, we present a detailed study of the CO rebinding kinetics in aqueous solution of a series of single- and double-site mutants of human myoglobin at positions 64, 68, 45, and 60. On the basis of the observed kinetics, we propose that the effect of surface residue 45 on the inner barrier can be explained by a chain of interactions between surface and pocket residues. Very large, and in some cases unexpected, changes are observed in the kinetics of recombination and in the partitioning between geminate and bimolecular recombination.

The kinetics of CO recombination to site-specific mutants of human myoglobin have been studied by flash photolysis in the temperature range 250-320 K on the nanosecond to second time scale in 75% glycerol at pH 7. The mutants were constructed to examine specific proposals concerning the roles of Lys 45, Asp 60, and Val 68 in the ligand binding process. It is found that ligand recombination is nonexponential for all the mutants and that both the geminate amplitude and rate show large variations. The results are interpreted in terms of specific models connecting the dynamics and structure. It is shown that removal of the charged group at position 45 does not substantially affect the barrier height for escape or entry of the ligand; therefore the breakage of the salt bridge linking Lys 45, Asp 60, and a heme propionate is ruled out as the rate-determining barrier for this process. On the other hand, it is found that the escape barrier decreases roughly as size of the residue at position 68 increases, in the order Ala > Val > Asn > Leu. The residue at position 68 is also a major contributor to the final barrier to rebinding, but the barrier height shows no correlation with residue size and is more dependent on the stereochemistry of the residue. A molecular mechanism for ligand binding that is consistent with the results is discussed, and supporting evidence for this mechanism is examined.

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