Linking conformation change to hemoglobin activation via chain-selective time-resolved resonance Raman spectroscopy of protoheme/mesoheme hybrids

Abstract

Time-resolved resonance Raman (RR) spectra are reported for hemoglobin (Hb) tetramers, in which the alpha and beta chains are selectively substituted with mesoheme. The Soret absorption band shift in mesoheme relative to protoheme permits chain-selective recording of heme RR spectra. The evolution of these spectra following HbCO photolysis shows that the geminate recombination rates and the yields are the same for the two chains, consistent with recent results on (15)N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxyheme nu (4) and nu (Fe-His) RR bands, which are anticorrelated. These shifts are resolved for the successive intermediates in the protein structure, which have previously been determined from time-resolved UV RR spectra. Both chains show Fe-His bond compression in the immediate photoproduct, which relaxes during the formation of the first intermediate, R(deoxy) (0.07 micros), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe-His bond weakens, more so for the alpha chains than for the beta chains. The weakening is gradual for the beta chains, but is abrupt for the alpha chains, coinciding with completion of the R-T quaternary transition, at 20 micros. Since the transition from fast- to slow-rebinding Hb also occurs at 20 micros, the drop in the alpha chain nu (Fe-His) supports the localization of ligation restraint to tension in the Fe-His bond, at least in the alpha chains. The mechanism is more complex in the beta chains.

Figure 1

Soret absorption envelops for the meso-proto hybrid Hb’s, showing the proximity of the 440 nm Raman laser wavelength to the deconvoluted proto-heme band.

Figure 2

The 440 nm - excited RR spectra of native (top) and meso-Hb (bottom), and of the two meso-proto hybrids. Band assignments and frequency shifts are indicated.

Figure 3

The RR spectra of hybrid Hb’s in the ν₄ region at the indicated delays following HbCO photolysis. The probe-only spectrum (bottom) reveals partial photolysis by the probe pulse.

Figure 4

The CO recombination progress curves for the indicated Hb forms, calculated via the ν₄ intensities.

Figure 5

Pump/probe minus probe only RR spectra at the indicated time delays. The ~228 cm⁻¹ ν_Fe-His bands in the probe only spectra (top) are due to partial photolysis. Static deoxy Hb spectra are compared (bottom).

Figure 6

Computed RR spectra in the low frequency region after HbCO photolysis, as determined from time-resolved UVRR analysis(18).

Figure 7

Computed RR spectra in the ν₄ region after HbCO photolysis, as determined from time-resolved UVRR analysis(18).

Figure 8

Allosteric model for the reaction path following HbCO photolysis showing the ν_Fe-His values for the intermediates (upper number for α subunit, lower number for βsubunit)[ see text for descriptions of the intermediates].

Figure 9

The ν_Fe-His/ν₄ correlation for the kinetic intermediates and for the deoxy Hb (T)