Analyzing the FMN—heme interdomain docking interactions in neuronal and inducible NOS isoforms by pulsed EPR experiments and conformational distribution modeling

Abstract

Nitric oxide synthases (NOSs), a family of flavo-hemoproteins with relatively rigid domains linked by flexible regions, require optimal FMN domain docking to the heme domain for efficient interdomain electron transfer (IET). To probe the FMN-heme interdomain docking, the magnetic dipole interactions between the FMN semiquinone radical (FMNH^•) and the low-spin ferric heme centers in oxygenase/FMN (oxyFMN) constructs of neuronal and inducible NOS (nNOS and iNOS, respectively) were measured using the relaxation-induced dipolar modulation enhancement (RIDME) technique. The FMNH^• RIDME data were analyzed using the mesoscale Monte Carlo calculations of conformational distributions of NOS, which were improved to account for the native degrees of freedom of the amino acid residues constituting the flexible interdomain tethers. This combined computational and experimental analysis allowed for the estimation of the stabilization energies and populations of the docking complexes of calmodulin (CaM) and the FMN domain with the heme domain. Moreover, combining the five-pulse and scaled four-pulse RIDME data into a single trace has significantly reduced the uncertainty in the estimated docking probabilities. The obtained FMN—heme domain docking energies for nNOS and iNOS were similar (-3.8 kcal/mol), in agreement with the high degree of conservation of the FMN—heme domain docking interface between the NOS isoforms. In spite of the similar energetics, the FMN—heme domain docking probabilities in nNOS and iNOS oxyFMN were noticeably different (~ 0.19 and 0.23, respectively), likely due to differences in the lengths of the FMN—heme interdomain tethers and the docking interface topographies. The analysis based on the IET theory and RIDME experiments indicates that the variations in conformational dynamics may account for half of the difference in the FMN—heme IET rates between the two NOS isoforms.

Graphical abstract