Structural evolution of the heme group during the allosteric transition in hemoglobin: Insights from resonance raman spectra of isotopically labeled heme

Abstract

Visible resonance Raman spectra in the low-frequency region (200–500 cm−1) are reported for hemoglobin (Hb) reconstituted with heme that is deuterated at the meso carbon atoms (meso-d4). Spectra were obtained in the deoxy form and in the immediate photoproduct of the carbonmonoxide adduct, HbCO. The isotope shifts permit assignment of two out-of-plane modes, γ6 and γ7, and the in-plane skeletal mode ν8, as well as the well-known iron-histidine [Fe-His] stretching vibration. Important differences between deoxyHb and the immediate photoproduct include 1) a large upshift in the Fe-His frequency, from 216 to 228 cm−1, 2) an upshift in γ6 (349 to 353 cm−1) together with substantial diminution of the ν8 (341 cm−1) intensity, and 3) collapse of two γ7 bands (305 and 296 cm−1) to a single band at 304 cm−1. This last observation implies subunit heterogeneity in deoxyHb but not in the photoproduct. When these bands are monitored in the time-resolved RR spectra following HbCO photolysis, it is seen that subunit heterogeneity is first detectable in the 0.5-μs transient [intermediate S], which has been associated with the initial rearrangement of the subunits to form the T-state contacts, on the basis of ultraviolet RR spectroscopy.1 However the intensification of ν8 does not occur until the 17-μs transient (intermediate T′), in which the T-state contacts are locked in and the Fe-His bond is strained. Implications for the mechanism of Hb allostery are discussed. © 1996 John Wiley & Sons, Inc.