Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase

Abstract

Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)­domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN–heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.

An Evaluation of Maximum Determination Methods for Center Line Slope Analysis

“…The remaining data were then fit to either a single Gaussian, Lorentzian, or pseudoVoigt function. Single Gaussians have also previously been used for CLS analysis in the literature …”

“…Single Gaussians have also previously been used for CLS analysis in the literature. 42 To attempt to extract maxima from pump slices without fitting, we also included the frequencies of maxima (referred to as the direct method) and the peak's center of mass from the raw data of pump slices. Using the center of mass requires the assumption that the peak is symmetric and unimodal, which is also an implicit assumption when fitting a portion of a pump slice to a single Gaussian, Lorentzian, or pseudoVoigt.…”