Figure 5. Reaction of ferrous His-Fe-His GLB-26 with CO or O₂.

GLB-26 was diluted in buffer saturated with CO: the slow absorption change in the visible region (560 nm minus 540 nm) is characteristic of the distal histidine replacement by CO (empty circles; the insert shows the variation of absorption between the spectra recorded few seconds after the mixing and after the completion of the replacement reaction). No such absorption change is observed by dilution into an oxygen saturated buffer (empty squares). Within a second after the mixing, faster than the time for His dissociation, deoxy GLB-26 is oxidized via electron transfer to the molecular O₂. This confirms the fast ms electron transfer by an outer-sphere mechanism to O₂ from ferrous His-Fe-His GLB-26, generated by CO photodissociation. Experimental conditions were 4 µM of globin in 25 mM potassium phosphate pH 7.0 at 8°C.