VIBRATIONAL ASSIGNMENTS OF THE FECO UNIT OF CO-BOUND HEME-PROTEINS REVISITED - OBSERVATION OF A NEW CO-ISOTOPE-SENSITIVE RAMAN BAND ASSIGNABLE TO THE FECO BENDING FUNDAMENTAL

In order to find the FeCO bending (delta(FeCO)) fundamental, resonance Raman spectra in the 600-200-cm(-1) region of CO adducts of hemoglobi n (Hb), its isolated chains, myoglobin (Mb), cytochrome c oxidase (CcO ), and cytochrome P-450 (P-450) were reexamined. A new CO-isotope-sens itive band was found around 365 cm(-1) for all but MbCO. For HbCO this band was located at 367, 355, 363, and 353 cm(-1) for (CO)-C-12-O-16, (CO)-C-13-O-16, (CO)-C-12-O-18, and (CO)-C-13-O-18 adducts, respectiv ely, and thus exhibited a zigzag pattern against the increase of the t otal mass of CO, similar to the 575-cm(-1) band which has been assigne d to the delta(FeCO) fundamental hitherto. Relative intensity of the s imilar to 365-cm(-1) band to the 575-cm(-1) band exhibited almost no c hange upon lowering of temperature from 20 to -10 degrees C, but since temperature dependent frequency shifts were appreciable, the present difference spectra could not determine if the intensity change by 11% expected for a difference combination band of a hot 210-cm(-1) mode wa s involved or not. The maximum intensity enhancement of the 365-cm(-1) band in the excitation profile within the Soret band occurred at long er wavelengths than that of the 575-cm(-1) band. The frequency differe nces between the CO-isotope-sensitive band around 575 cm(-1) and one a round 365 cm(-1) were 208 +/- 3, 213 +/- 5, and 204 +/- 3 cm(-1) for a ll CO isotopes of HbCO, CcO CO, and P-450 CO, respectively. Despite th at the Fe-CO and C-O stretching modes exhibited no deuteration shifts, the frequency of the 575-cm(-1) band was higher in D2O than in H2O, a nd this feature was more prominent with MbCO than HbCO. The 575-cm(-1) band of MbCO was remarkably strong upon excitation at 406.7 nm, and t his is distinct from other proteins examined. These observations sugge st that the similar to 365-cm(-1) band arises from the delta(FeCO) fun damental and the 575-cm(-1) band is its combination with a porphyrin v ibration or possibly a Fe-C deformation mode. Normal-coordinate calcul ations for the isolated FeCO unit reasonably reproduce the observed is otopic frequency shifts under these assignments.