Resonance Raman studies of hemoglobin structural dynamics

Cynthia Rajani, Marquette University


In an effort to resolve an existing cintroversy involving the assignment of the bending fundamental, $\delta$(FeCO), for CO-ligated heme proteins, resonance Raman studies of hemoglobin (Hb), containing hemes that are selectively deuterated, as well, as, native CO-Hb were carried out. Several CO isotope sensitive modes were observed in the low frequency region when difference spectra were generated between natural abundance and doubly labelled CO isotopomers. These features were shown to arise from small (1-2 cm$\sp{-1}$) shifts of one or two heme macrocycle modes. In the frequency region between 700 and 1200 cm$\sp{-1}$, the CO-isotope sensitive features previously ascribed to overtones and combinations involving an $\delta$(FeCO) mode at $\sim$370 cm$\sp{-1}$ were instead attributed to combinations of low frequency heme modes with the earlier assignment of $\delta$(FeCO) at 578 cm$\sp{-1}$ or with $\nu$(FeC) at 506 cm$\sp{-1}$. There are several key marker modes of the heme macrocycle which are sensitive to methine bridge deuteration, exhibiting shifts of up to 25 cm$\sp{-1}$. Hb hybrids in which only one type of subunit, $\alpha$ or $\beta$, contains a deuterated heme were made, thus allowing spectral resolution of the two subunits within an intact Hb tetramer Transient studies revealed that in the hybrid containing deuterated $\alpha$-subunits, a core-marker band, $\nu\sb{19}$, occurred $\sim$2 cm$\sp{-1}$ higher in the deoxy form than in the photoproduct at 10 ns. The $\nu\sb{19}$ mode for the $\beta$-subunit containing native heme experienced no shift. These results were confirmed in the alternate hybrid, wherein the $\beta$-subunit contained the deuterated heme. Time-resolved resonance Raman studies were performed on both the native Hb tetramer and on the hybrids mentioned above. The time resolution of the $\nu\sb{19}$ mode was examined and complete relaxation after photolysis to the deoxy value occurred between 216-350 ns. The re-ligation dynamics also were examined in the geminate phase (20-800 ns after photolysis) and at 20 ns, geminate ($\sim$50%) recombination was complete.

Recommended Citation

Rajani, Cynthia, "Resonance Raman studies of hemoglobin structural dynamics" (1998). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI9901736.