Probing Heme Active Sites of Hemoglobin in Functional Red Blood Cells Using Resonance Raman Spectroscopy

The UV-vis absorption, Raman imaging, and resonance Raman (rR) spectroscopy methods were employed to study cyanohemoglobin (HbCN) adducts inside living functional red blood cells (RBCs). The cyanide ligands are especially optically sensitive probes of the active site environment of heme proteins. The rR studies of HbCN and its isotopic analogues (¹³CN^-, C¹⁵N^-, and ¹³C¹⁵N^-), as well as a careful deconvolution of spectral data, revealed that the ν(Fe-CN) stretching, δ(Fe-CN) bending, and ν(C≡N) stretching modes occur at 454, 382, and 2123 cm^-1, respectively. Interestingly, while the ν(Fe-CN) modes exhibit the same frequencies in both the isolated and RBC-enclosed hemoglobin molecules, small frequency differences are observed in the δ(Fe-CN) bending modes and the values of their isotopic shifts. These studies show that even though the overall tilted conformation of the Fe-C≡N fragment in the isolated HbCN is preserved in the HbCN enclosed within living cells, there is a small difference in the degree of distortion of the Fe-C≡N fragment. The slight changes in the ligand geometry can be reasonably attributed to the high ordering and tight packing of Hb molecules inside RBCs.