Resonance Raman studies of selectively labelled hemoglobin tetramers

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-d(4)), methyl groups (oxyHb-d(12)), and both meso pos itions and methyl groups (oxyHb-d(16)). Analyzed collectively, the RR spect ra of the low-spin dioxygen adduct species studied here reveal isotopic-sen sitive modes that induce subtle differences in shape of the spectrum of oxy hemoglobins. The most significant spectral differences are observed in the region of 350-440 cm(-1) which contains bending modes of the peripheral sub stituents, i.e. delta (C13,17CcCd) and delta (CCalphaCbeta) + delta (CN) (s tructure and atom numbering scheme being given in Fig. 1). Several in-plane (v(9), v25, v8, v50, v33, v25 and v48) in-plane (v(9), v(25), v(8), v(50), v(33), v(25) and v(48)) and out-of plane (gamma (7), gamma (16), gamma (22 ), and gamma (21)) heme vibrations have also been identified. The results p resented here provide convincing evidence for the utility of selectively la belled hemoglobins in the definitive assignment of the low-frequency Raman bands. (C) 2001 Elsevier Science B.V. All rights reserved.