UV Resonance Raman Determination of α-Helix Melting During the Acid Denaturation of Myoglobin

Abstract

We have studied the absorption spectrum and the 206.5 and 229 mn excited resonance Raman spectrum of aquometmyoglobin (Mb) between pH 7.5 and pH 1.5. The acid denaturation of Mb below pH 3.5 results in small absorption increases below 220 nm due to α-helix melting, and an absorption decrease at ~230 nm due to environmental changes of the trp and/or tyr residues. Dramatic heme Soret band changes indicate major alterations of the helve pocket. We examined the 206.5 nm resonance Raman spectra, which selectively enhances amide backbone vibrations and compared these results to the 229 nm excited resonance Raman spectra, which selectively enhances tyr and trp vibrations. We calculate that the Mb α-helical composition decreases from ∼80% at neutral pH to ∼19% below pH 3.5. The trp Raman cross sections dramatically decrease at low pH which indicates that the A helix melts. The tyr Raman bands are pH independent which indicates that the G and H helices do not melt. This result indicates that helices A, B, C, D and E melt in a concerted fashion, while the antiparallel G and H helices only partially melt. This melting of the helices framing the heme pocket is responsible for the change in heme binding; the diffuseness of the unfolded Mb heme Soret band suggests that the heme no longer has a single defined binding site.