Selective X-ray induced NO-photodissociation in hemoglobin crystals: evidences from a Raman assisted-crystallographic study

Despite the high physiological relevance, hemoglobin crystal structures with NO bound to heme constitute less than 1 % of the total ligated hemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO ligated Hbs is most likely related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O2. Here, using Raman assisted-X-ray crystallography, we show that under X-ray exposure (at four different radiation doses), crystals of nitrosylated hemoglobin from Trematomus bernacchii undergo a transition, mainly at the β chains, generating a pentacoordinate species, due to photodissociation of the Fe-NO bond. These data give a physical explanation of the low content of nitrosylated Hb structures available in the literature and provide a rough estimate of the relative Raman cross-section of bands corresponding to deoxygenated and nitrosylated hemes.