Resonance Raman studies of hemoglobin with selectively deuterated hemes. A new perspective on the controversial assignment of the Fe-CO bending mode

In an effort to resolve an existing controversy involving the assignment of the bending fundamental [delta(FeCO)] of CO-ligated heme proteins, resonance Raman studies of the CO adducts of native hemoglobin (Hb), together with Hb containing selectively deuterated hemes, have been conducted. Hemes were utilized which were deuterated at the peripheral methyl positions (d12) or at the methine carbon positions (d4). Several CO isotope sensitive modes were observed in the low-frequency region (300-400 cm(-1)) by generating difference spectra from the absolute spectra of the natural abundance (NA) and doubly labeled (CO)-C-13-O-18 (DI) CO-Hb. These features included one at similar to 370 cm(-1). Deconvolution of the low-frequency region revealed that a 1-2 cm(-1) shift of a heme mode at 367 cm(-1) is responsible for this difference feature. In the mid-frequency spectral region (650-1300 cm(-1)), features which were previously suggested to be ascribable to combination bands and overtones involving the proposed similar to 370 cm(-1) delta(FeCO) fundamental showed sensitivity to heme deuteration, a fact which suggests that these modes are combinations involving heme modes rather than two internal fundamentals of the FeCO fragment. These results imply, therefore, that the observation of weak isotope-sensitive features in the 700-900 cm(-1) region does not support the assignment of the similar to 370 cm(-1) feature to the delta(FeCO) fundamental, but these features are more reasonably interpreted to arise from combinations involving the relatively strong v(Fe-CO) and lower frequency heme modes, an interpretation that is consistent with the long-standing assignment of the weak band near 580 cm(-1) to the delta(FeCO) fundamental.