Endothelial nitric oxide synthase (eNOS) is a membrane-anchored enzyme. To highlight the potential role and effect of membrane phospholipids on the structure and activity of eNOS, we have incorporated the recombinant oxygenase subunit of eNOS into lipid nanodiscs. Two different size distribution modes were detected by multi-angle dynamic light scattering both for empty nanodiscs, and nanodiscs-bound eNOS(oxy). The calculated hydrodynamic diameter for mode 1 species was 9.0 nm for empty nanodiscs and 9.8 nm for nanodisc bound eNOS(oxy). Spectroscopic Griess assay was used to measure the enzymatic activity. Remarkably, the specific activity of nanodisc-bound eNOS(oxy) is similar to 65% lower than the activity of free enzyme. The data shows that the nano-membrane environment affects the catalytic properties of eNOS heme domain. (C) 2017 Elsevier Inc. All rights reserved.