Structure-function paradigm in Human Myoglobin: how a single-residue substitution affects NO reactivity at low pO2.

This work is focused on the two more expressed human myoglobin isoforms. In the literature, their different over-expression in high-altitude natives was proposed to be related to alternative/complementary functions in hypoxia. Interestingly, they differ only at residue-54, lysine or glutamate, which is external and far from the main binding site. In order to ascertain whether these two almost identical myoglobins might exert different functions and to contribute to a deeper understanding about myoglobin's oxygen-level dependent functioning, they have been compared with respect to dynamics, heme electronic structure and NO reactivity at different O2 levels. Electron paramagnetic resonance spectroscopy was employed to investigate the electronic structure of the nitrosyl-form, obtaining fundamental clues about a different bond-interaction between the heme-iron and the proximal histidine, and highlighting striking differences in NO reactivity, especially at a very low pO2. The experimental results well matched with the information provided by molecular dynamics simulations, which showed a significantly different dynamics for the two proteins only in the absence of O2. The single mutation differentiating the two myoglobins resulted to strongly affect the plasticity of the CD region (C-helix - loop - D-helix), whose fluctuations, being coupled to the solvent, were found to be correlated with the dynamics of the distal binding site. In the absence of O2, on the one hand a significantly different probability for the histidine-gate opening has been shown by MD simulations, on the other a different yield of myoglobin-NO formation was experimentally observed through EPR.