Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

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

PNAS 2013 110 (38) E3577-E3586; published ahead of print September 3, 2013,doi:10.1073/pnas.1313331110

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Significance

The biological role of nitric oxide (NO) in mammalian physiology is now well established as a signaling molecule in the cardiovascular and nervous systems and as a chemical component in the host response to infection. NO is synthesized by the enzyme NO synthase (NOS). The structure of the entire NOS enzyme has not been solved, but the structure of isolated domains has been reported. In this study, we use a mass spectrometry approach (hydrogen–deuterium exchange) to find interaction surfaces of the native protein. These results were then used to generate NOS models, which revealed interaction surfaces that mediate NOS activity. These interacting surfaces provide insight into the conformational changes and residues necessary for regulating NOS activity.

Abstract

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.