PROTEIN RESPONSE TO PHOTODISSOCIATION OF CO FROM CARBONMONOXYMYOGLOBIN PROBED BY TIME-RESOLVED INFRARED-SPECTROSCOPY OF THE AMIDE-I BAND

Protein conformational changes coupled to the ligation reactions of ca rbon monoxide in myoglobin (Mb) are detected by time-resolved infrared spectroscopy. An apparatus based on a tunable diode laser operating i n the region of 1650 cm-1 is used to probe changes in the amide I abso rption band of the protein in response to photodissociation and subseq uent rebinding of CO. The time course of changes in the amide I band i s shown to follow the recombination of photolyzed CO with Mb. A time-r esolved difference spectrum in the amide I region is generated by tuni ng the diode laser probe source. The features in the IR difference spe ctrum are assigned to the motions of the polypeptide backbone associat ed with the global relaxation of the protein from the ligated to the d eoxy conformation. A static difference spectrum generated by subtracti ng FTIR spectra of carbonmonoxy-Mb and deoxy-Mb is essentially identic al to the transient spectrum, indicating that the protein relaxation i s complete with the 100-ns time resolution of the experiment.