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Structural studies of constitutive nitric oxide synthases with diatomic ligands bound

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Abstract

Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)–arginine–CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with l-arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY}n formalism developed from the inorganic diatomic–metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O2) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.

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Abbreviations

bsNOS:

NOS-like protein from Bacillus subtilis

CaM:

Calmodulin

eNOS:

Endothelial NOS

EPR:

Electron paramagnetic resonance

FMN:

Flavin mononucleotide

HEPES:

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

H2B:

7,8-Dihydrobiopterin

H4B:

(6R)-5,6,7,8-Tetrahydrobiopterin

iNOS:

Inducible NOS

MES:

2-Morpholinoethanesulfonic acid

NHA:

N ω-Hydroxy-l-arginine

nNOS:

Neuronal NOS

NOS:

Nitric oxide synthase

NSD:

Normal structural deformation

PEG3350:

Poly(ethylene glycol) 3350

PMSF:

Phenylmethanesulfonyl fluoride

Tris:

Tris(hydroxymethyl)aminomethane

βME:

2-Mercaptoethanol

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Acknowledgements

This research was supported by NIH grant GM57353. We thank Bettie Sue Masters and Pavel Martasek (University of Texas Health Science Center, San Antonio, USA) for the eNOS heme domain used is this study and C.S. Raman (University of Texas Health Science Center, Houston, USA) for his efforts in helping to determine the eNOS-CO complex structure. We thank Dmitry Pervisky for setting up an NO transfer line and also thank the beamline staff at the Advanced Light Source and Stanford Synchrotron Radiation Laboratory for assistance during data collection.

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Authors and Affiliations

  1. Departments of Molecular Biology and Biochemistry, Chemistry, and Physiology and Biophysics and the Center in Chemical and Structural Biology, University of California, Irvine, CA, 92697-3900, USA

    Huiying Li, Jotaro Igarashi, Joumana Jamal, Weiping Yang & Thomas L. Poulos

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  1. Huiying Li
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  2. Jotaro Igarashi
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  3. Joumana Jamal
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Correspondence to Thomas L. Poulos.

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Li, H., Igarashi, J., Jamal, J. et al. Structural studies of constitutive nitric oxide synthases with diatomic ligands bound. J Biol Inorg Chem 11, 753–768 (2006). https://doi.org/10.1007/s00775-006-0123-8

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