Intersubunit Communication via Changes in Hemoglobin Quaternary Structures Revealed by Time-Resolved Resonance Raman Spectroscopy: Direct Observation of the Perutz Mechanism

Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the beta subunits. Changes in the resonance Raman spectra of the alpha heme and the beta Ni-heme groups in the hybrid hemoglobin were observed upon CO photolysis in the alpha subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the upsilon(Fe-His) and the gamma(7) band of alpha heme was similar to that of unsubstituted hemoglobin, suggesting that substitution with Ni-heme did not perturb the allosteric dynamics of the hemoglobin. In the beta subunits, no structural change in the Ni-heme was observed until 1 mu s. In the microsecond regime, temporal evolution of the frequencies of the upsilon(Ni-His) and the gamma(7) band of beta Ni heme was observed concomitant with an R T quaternary change at about 20 The changes in the upsilon(Fe-His) and upsilon(Ni-His) frequencies of the a and beta subunits with the common time constant of similar to 20 mu s indicated that the proximal tension imposed on the bond between the heme and the proximal histidine strengthened upon the quaternary changes in both the alpha and the beta subunits concertedly. This observation is consistent with the Perutz mechanism for allosteric control of oxygen binding in hemoglobin and, thus, is the first real-time observation of the mechanism. Protein dynamics and allostery based on the observed time-resolved spectra also are discussed.