Besides the well known role of O₂ storage/delivery system, Myoglobin (Mb) has been reported to maintain cellular respiration at the optimum by nitric oxide (NO) scavenging, an inhibitor of cytochrome c oxidase.

Among the five Human Mb isoforms, the most expressed, Mb I and Mb II, differ for a single residue (54 K vs E, respectively). Tibetan population, a unique model to investigate adaption to hypoxia, present a high systemic blood flow with high levels of circulating biologically active NO metabolites and are characterized by a significant increase of Mb II. A cause-effect relationship between the isoform expression and an evolutionary response to the hypoxic high-altitude environment was hypothesized. Electron paramagnetic resonance (EPR) was used to compare the NO binding capacity of MbI and MbII isoforms, at different [O₂] (0, 4, 21%), related to structural differences too.

The proteins (0.05 mM) were incubated (37°C, 30 min) in the presence of the NO donor (50 mM) at the chosen PO₂. EPR spectra were recorded at 77 K by an E-SCAN (Bruker) instrument operating at X band. NOMb concentrations (mM) were obtained from a calibration procedure.

In the deoxy-form, an almost three times greater NO binding capacity of Mb II vs Mb I was calculated. The difference disappeared increasing the PO2. EPR results were supported by Molecular Dynamics simulation data: deoxy-MbII showed higher probability for distal pocket piston-like movements, almost vanishing in the oxy-form.