Resonance Raman studies of selectively labelled hemoglobin tetramers

Abstract

In this paper, we report the high quality low-frequency resonance Raman (RR) spectra of oxyhemoglobin (oxyHb) and its reconstituted analogs, in which protons in ferric protoporphyrin IX were substituted by deuterium atoms in meso positions (oxyHb-d4), methyl groups (oxyHb-d12), and both meso positions and methyl groups (oxyHb-d16). Analyzed collectively, the RR spectra of the low-spin dioxygen adduct species studied here reveal isotopic-sensitive modes that induce subtle differences in shape of the spectrum of oxyhemoglobins. The most significant spectral differences are observed in the region of 350–440cm−1 which contains bending modes of the peripheral substituents, i.e. δ(C13,17CcCd) and δ(CCαCβ)+δ(CN) (structure and atom numbering scheme being given in Fig. 1) . Several in-plane (ν9, ν25, ν8, ν50, ν33, ν25 and ν48) and out-of-plane (γ7, γ16, γ22, and γ21) heme vibrations have also been identified. The results presented here provide convincing evidence for the utility of selectively labelled hemoglobins in the definitive assignment of the low-frequency Raman bands.