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 stretching band at 1951 cm-1 serves as a convenient monitor of the state of ligation. Both geminate and second-order phases of CO recombination occur at rates which are in excellent agreement with previous visible absorption measurements, showing the molecular mechanisms to be unperturbed by the high protein concentrations (6.7 mM in heme) required for adequate protein signals. While the extent of photolysis (43%) was insufficient to drive the R-->T quaternary transition, the protein TRIR (time-resolved infrared) difference bands (1250-1700 cm-1) nevertheless reveal interesting tertiary dynamics. Most of the bands are fully developed at very early times, possibly preceding the geminate recombination phase (tau = 50 ns). Some bands arise more slowly, however, with a time constant of 0.4 microsecond, reflecting a tertiary motion which is coincident with a quaternary motion previously detected by ultraviolet resonance Raman spectroscopy of fully photolyzed HbCO. Relaxation of the TRIR bands is either faster (tau = approximately 90 microseconds) or slower (tau = approximately 250 microseconds) than CO rebinding (effective time constant of 160 microseconds), suggesting either a distribution of tertiary processes or a chain inequivalence in CO rebinding.
Hu
X
X
Department of Chemistry, Princeton University, New Jersey 08544, USA.
Frei
H
H
Spiro
T G
TG
D002248:Carbon Monoxide / Q000378:metabolism; D002263:Carboxyhemoglobin / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D010782:Photolysis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared
D006454:Hemoglobins; D008024:Ligands; D006418:Heme; D002248:Carbon Monoxide; D002263:Carboxyhemoglobin