Journal Article

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

Eric S. Underbakke, William R. Schief, Daniel W. Kulp, Brian C. Smith, Michael A. Marletta

2013

Overview

Nitric oxide (NO) produced by NO synthase (NOS) participates in diverse physiological processes such as vasodilation, neurotransmission, and the innate immune response. Mammalian NOS isoforms are homodimers composed of two domains connected by an intervening calmodulin-binding region. The N-terminal oxidase domain binds heme and tetrahydrobiopterin and the arginine substrate. The C-terminal reductase domain binds FAD and FMN and the cosubstrate NADPH. Although several high-resolution structures of individual NOS domains have been reported, a structure of a NOS holoenzyme has remained elusive. Determination of the higher-order domain architecture of NOS is essential to elucidate the molecular underpinnings of NO formation. In particular, the pathway of electron transfer from FMN to heme, and the mechanism through which calmodulin activates this electron transfer, are largely unknown. In this report, hydrogen–deuterium exchange mass spectrometry was used to map critical NOS interaction surfaces. Direct interactions between the heme domain, the FMN subdomain, and calmodulin were observed. These interaction surfaces were confirmed by kinetic studies of site-specific interface mutants. Integration of the hydrogen–deuterium exchange mass spectrometry results with computational docking resulted in models of the NOS heme and FMN subdomain bound to calmodulin. These models suggest a pathway for electron transfer from FMN to heme and a mechanism for calmodulin activation of this critical step.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Animals / arginine / Biological Sciences / calmodulin / Calmodulin - chemistry / Calmodulin - metabolism / Deuterium / Deuterium Exchange Measurement / Dimerization / Electron transfer / Electron Transport / Electrophoresis, Polyacrylamide Gel / Flavin Mononucleotide - metabolism / Flavin-Adenine Dinucleotide - metabolism / Fluorescence / heme / Heme - metabolism / Immune response / innate immunity / Kinetics / mammals / Mass Spectrometry / Models, Molecular / mutants / Mutation / NADP (coenzyme) / Nitric oxide / nitric oxide synthase / Nitric Oxide Synthase Type II - chemistry / Nitric Oxide Synthase Type II - genetics / Nitric Oxide Synthase Type II - metabolism / PNAS Plus / Protein Conformation / Species Specificity / vasodilation