Abstract
Nitric oxide synthase (NOS) is an enzyme responsible for the production of nitric oxide in living organisms. Structurally, it is a homodimer composed of multiple domains connected by random coil tethers. The resulting structural flexibility, along with the diverse conformational states it enables, is essential for NOS function and remains an active area of investigations. Here, we studied the docking interactions between the reductase domains of NOS subunits. To probe these interactions, a nitroxide-based bifunctional spin label was attached to each T34C/S38C calmodulin (CaM) molecule bound to the CaM-binding region of the tether, which connects the oxygenase and flavin mononucleotide (FMN) domains in each subunit of the homodimeric oxygenase/FMN (oxyFMN) construct of rat neuronal NOS (nNOS). The magnetic dipole interaction between the spin labels was detected by 2 + 1 electron spin echo (ESE) methods. The experimental 2 + 1 ESE traces were interpreted using the Monte Carlo calculations of NOS conformational distributions. The results unequivocally show that at the estimated effective temperature of the frozen conformational distribution, T(ef) ≈ 200 K, a large proportion of the oxyFMN proteins (~ 55%) adopt a clamp-shaped conformation in which the FMN domains of different NOS subunits dock with each other. The stabilization energy of this docking complex (i.e., docking energy) was estimated in the model of isotropic interaction as - 7.2kT(ef) ≈ - 2.9 kcal/mol. The identification of this clamp-shaped conformation suggests it as an intermediate structural state that may influence NOS catalytic efficiency by facilitating the FMN-heme interdomain electron transfer through constraining the conformational space accessible to the FMN domain as it approaches its docking positions at the heme domain.