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 assignme nt of the bending fundamental [delta(FeCO)] of CO-ligated heme protein s, 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). Sever al CO isotope sensitive modes were observed in the low-frequency regio n (300-400 cm(-1)) by generating difference spectra from the absolute spectra of the natural abundance (NA) and doubly labeled (CO)-C-13-O-1 8 (DI) CO-Hb. These features included one at similar to 370 cm(-1). De convolution of the low-frequency region revealed that a 1-2 cm(-1) shi ft of a heme mode at 367 cm(-1) is responsible for this difference fea ture. 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 sug gests that these modes are combinations involving heme modes rather th an two internal fundamentals of the FeCO fragment. These results imply , therefore, that the observation of weak isotope-sensitive features i n the 700-900 cm(-1) region does not support the assignment of the sim ilar to 370 cm(-1) feature to the delta(FeCO) fundamental, but these f eatures are more reasonably interpreted to arise from combinations inv olving 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.