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 hemoglobin (Hb), its isolated chains, myoglobin (Mb), cytochrome c oxidase (CcO), and cytochrome P-450 (P-450) were reexamined. A new CO-isotope-sensitive 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, respectively, and thus exhibited a zigzag pattern against the increase of the total mass of CO, similar to the 575-cm(-1) band which has been assigned to the delta(FeCO) fundamental hitherto. Relative intensity of the similar to 365-cm(-1) band to the 575-cm(-1) band exhibited almost no change 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 was involved or not. The maximum intensity enhancement of the 365-cm(-1) band in the excitation profile within the Soret band occurred at longer wavelengths than that of the 575-cm(-1) band. The frequency differences between the CO-isotope-sensitive band around 575 cm(-1) and one around 365 cm(-1) were 208 +/- 3, 213 +/- 5, and 204 +/- 3 cm(-1) for all CO isotopes of HbCO, CcO CO, and P-450 CO, respectively. Despite that 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, and 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 this is distinct from other proteins examined. These observations suggest that the similar to 365-cm(-1) band arises from the delta(FeCO) fundamental and the 575-cm(-1) band is its combination with a porphyrin vibration or possibly a Fe-C deformation mode. Normal-coordinate calculations for the isolated FeCO unit reasonably reproduce the observed isotopic frequency shifts under these assignments.