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

Abstract

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 β subunits. Changes in the resonance Raman spectra of the α heme and the β Ni-heme groups in the hybrid hemoglobin were observed upon CO photolysis in the α subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the ν(Fe-His) and the γ7 band of α 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 β subunits, no structural change in the Ni-heme was observed until 1 μs. In the microsecond regime, temporal evolution of the frequencies of the ν(Ni-His) and the γ7 band of β Ni-heme was observed concomitant with an R → T quaternary change at about 20 μs. The changes in the ν(Fe-His) and ν(Ni-His) frequencies of the α and β subunits with the common time constant of ∼20 μs indicated that the proximal tension imposed on the bond between the heme and the proximal histidine strengthened upon the quaternary changes in both the α and the β 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.