Structural Basis for Isozyme-specific Regulation of Electron Transfer in Nitric-oxide Synthase

Elsa D Garcin,Christopher M Bruns,Sarah J Lloyd,David J Hosfield,Mauro Tiso,Ratan Gachhui,Dennis J Stuehr,John A Tainer,Elizabeth D Getzoff

doi:10.1074/jbc.m406204200

Elsa D Garcin, Christopher M Bruns + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m406204200

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Abstract

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

Highlights

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2؉/calmodulin-triggered Nitric oxide (NO) synthesis
Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire flavin mononucleotide (FMN) domain to deliver electrons to the catalytic module in the holoenzyme
We used the structure of CYPOR [13], without its FMN domain, as a starting model for molecular replacement and manually built the nNOS FMN

Summary

Introduction

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2؉/calmodulin-triggered NO synthesis. We address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electronsupplying, neuronal NOS reductase dimer By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. NOSred belongs to a large protein family that includes NADPH-dependent cytochrome P450 reductase (CYPOR), sulfite reductase flavoprotein and novel reductase 1 These reductases share a conserved organization of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH)-binding domains [13,14,15]. Electron transfer proceeds from NADPH to FAD to FMN to heme [18] This last step is rate-limiting [19], occurs in trans from NOSred of one polypeptide to NOSox of the other, and is uniquely triggered by conformational changes induced by Ca2ϩ/CaM binding [20]

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