Mutation in the FMN Domain Modulates MCD Spectra of the iNOS Ferric Cyano Complex in a Substrate-Specific Manner

摘要

We have obtained low temperature MCD spectra for ferric cyano complexes of the wild type and E546N mutant of a human iNOS oxygenase/FMN construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the L-arginine-bound ferric iNOS heme [Sempombe et al., J. Am. Chem. Soc. 2009, 131, 6940–6941]. Addition of L-arginine to the wild type protein causes notable changes in the CN⁻-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with L-arginine is absent in the CN⁻ complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. EPR spectroscopy also reveals a substrate dependency of the spectra. These results indicate that the interdomain FMN–heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.