Effect of the N-terminal residues on the quaternary dynamics of human adult hemoglobin

- We examined time-resolved Raman spectra of two kinds of recombinant hemoglobin.
- A frequency shift of ν(Fe-His) band indicated tertiary and quaternary changes.
- Spectral changes in the tens of microseconds region were affected by the N-terminus.
- An N-terminus changes in the second step of the quaternary structure change.

The protein dynamics of human hemoglobin following ligand photolysis was studied by time-resolved resonance Raman spectroscopy. The time-resolved spectra of two kinds of recombinant hemoglobin expressed in Escherichia coli, normal recombinant hemoglobin and the α(V1M)/β(V1M) double mutant, were compared with those of human adult hemoglobin (HbA) purified from blood. A frequency shift of the iron-histidine stretching [ν(Fe-His)] band was observed in the time-resolved spectra of all three hemoglobin samples, indicative of tertiary and quaternary changes in the protein following photolysis. The spectral changes of the α(V1M)/β(V1M) double mutant were distinct from those of HbA in the tens of microseconds region, whereas the spectral changes of normal recombinant hemoglobin were similar to those of HbA isolated from blood. These results demonstrated that a structural change in the N-termini is involved in the second step of the quaternary structure change of hemoglobin. We discuss the implications of these results for understanding the allosteric pathway of HbA.

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