We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10-8 to 10-1 s by flash photolysis, in which the photodissociated CO rebinds to the heine iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64→Val/Val68→Thr (H64V/V68T) and His64→Val/Val68→Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5°C, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.
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