CO recombination to human myoglobin mutants in glycerol-water solutions.

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.