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 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.
Department of Chemistry, Graduate School of Science, Osaka University , 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
Kenta
K
Yamada
Haruto
H
Ishikawa
Misao
M
Mizuno
Naoya
N
Shibayama
Yasuhisa
Y
Mizutani
D002248:Carbon Monoxide / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D009532:Nickel / Q000737:chemistry; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D013997:Time Factors
D006454:Hemoglobins; D021122:Protein Subunits; D006418:Heme; D006639:Histidine; D009532:Nickel; D002248:Carbon Monoxide; D007501:Iron