A combined Raman microspectroscopic / crystallographic approach to photolytic and oxidative denitrosylation in hemoglobins

Despite the high physiological relevance, hemoglobin crystal structures with NO bound to heme are less than 1 % of the total ligated hemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO ligated Hbs is probably related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O2. We investigated the oxidative denitrosylation of the hemoglobin from Trematomus bernacchii (HbTb) via Raman-assisted crystallography. Furthermore, using X-ray crystallography-assisted Raman microscopy at the Swiss Light Source [1], we show that under X-ray exposure (at four different radiation doses), crystals of nitrosylated HbTb undergo a selective transition at the β chains generating a pentacoordinate species, due to photodissociation of the Fe-NO bond. This phenomenon was recently observed also in HbA solutions [2]. These observations give a physical explanation of the low content of nitrosylated Hb structures available in the literature and provide a valuable quantitative estimate of the relative Raman cross-section of bands corresponding to deoxygenated and nitrosylated hemes. The assembly of structures at different nitrosylation states provides a wide snapshot of the communication mechanism between α and β subunits of deoxygenated HbTb upon undergoing nitrosylation[3]. PNRA is acknowledged for financial support.