Monitoring the Allosteric Transition and CO Rebinding in Hemoglobin with Time-Resolved FTIR Spectroscopy

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Time-resolved FTIR spectra are reported for the photocycle of carbonmonoxy hemoglobin, under saturating photolysis conditions, which are sufficient to drive the R−T allosteric transition. Direct evidence for this transition was provided by the microsecond time scale evolution of an 1857 cm-1 bisignate difference band (cysteine S−H stretching), which is a marker of the T state. The time course of the strong 1951 cm-1 band of bound CO showed the expected fast geminate and slower second-order rebinding phases. Two slow phases were observed, having time constants consistent with reported binding rates for R and T state molecules. The geminate yield was 50%, the majority (37%) rebinding with a 70 ns time constant, consistent with previous studies, but an additional low-amplitude (13%) phase was resolved, with an 890 ns time-constant. Difference FTIR bands are also observed in the 1300−1700 cm-1 region, where protein vibrations are expected. In the nanosecond regime these bands varied irregularly, due to instrument limitations, but in the microsecond regime they evolved (30 μs time constant) toward the static difference spectrum of HbCO minus deoxyHb, reflecting the expected evolution from R to T state photoproduct molecules. The difference spectra of R and T photoproduct molecules extracted from the data via kinetic analysis contain not only common bands but also bands that are distinctive. The R photoproduct difference spectrum contains a positive/negative band pair at 1649 and 1683 cm-1, which is interpreted as resulting from the breaking of one or more α-helical carbonyl H-bonds. Candidate H-bonds are those that connect the H-helix residues Tyr α140 and β145 with the F-helix residues Val α93 and β95, in both HbCO and deoxyHb. These H-bonds are believed to break and reform at intermediate stages of the allosteric pathway, on the basis of UV Raman evidence.