NANOSECOND STEP-SCAN FTIR SPECTROSCOPY OF HEMOGLOBIN - LIGAND RECOMBINATION AND PROTEIN CONFORMATIONAL-CHANGES

Step-scan FTIR spectroscopy with nanosecond time resolution is applied to the photocycle of carbonmonoxy hemoglobin (HbCO). The strong CO st retching band at 1951 cm(-1) serves as a convenient monitor of the sta te of ligation. Both geminate and second-order phases of CO recombinat ion occur at rates which are in excellent agreement with previous visi ble absorption measurements, showing the molecular mechanisms to be un perturbed by the high protein concentrations (6.7 mM in heme) required for adequate protein signals. While the extent of photolysis (43%) wa s insufficient to drive the R-->T quaternary transition, the protein T RIR (time-resolved infrared) difference bands (1250-1700 cm(-1)) never theless reveal interesting tertiary dynamics. Most of the bands are fu lly developed at very early times, possibly preceding the geminate rec ombination phase (tau = 50 ns). Some bands arise more slowly, however, with a time constant of 0.4 mu s, reflecting a tertiary motion which is coincident with a quaternary motion previously detected by ultravio let resonance Raman spectroscopy of fully photolyzed HbCO. Relaxation of the TRIP bands is either faster (tau = similar to 90 mu s) or slowe r (tau = similar to 250 mu s) than CO rebinding (effective time consta nt of 160 mu s), ring either a distribution of tertiary processes or a chain inequivalence in CO rebinding.