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Substrate and substrate analog binding to endothelial nitric oxide synthase: electron paramagnetic resonance as an isoform-specific probe of the binding mode of substrate analogs.
Biochemistry. 1997 Sep 30; 36(39):11821-7.B

Abstract

The binding of arginine analogs to endothelial nitric oxide synthase (eNOS, NOSIII) perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Using electron paramagnetic resonance as a probe of heme ligation geometry, four categories of high-spin complex could be distinguished. These are analogous to the four classes of high-spin complexes, stabilized individually by the binding of L-arginine, N-hydroxy-L-arginine (NHA), N-methyl-L-arginine (NMA), and N-nitro-L-arginine (NNA), which we have previously reported for the other two isoforms. Each of these species is five-coordinate and retains the axial thiolate ligand but each differs in its ligation geometry. N-Methyl-L-arginine is a relatively poor inhibitor of eNOS, and the NMA complex of eNOS differs from the N-methyl-L-arginine complexes of inducible nitric oxide synthase (iNOS, NOSII) and neuronal nitric oxide synthase (nNOS, NOSI) in that it is of lower rhombicity. We previously showed that inducible nitric oxide synthase, which binds NNA less tightly than eNOS and nNOS, could not form the lower rhombicity NNA complex characteristic of nNOS. Endothelial nitric oxide synthase readily forms such lower rhombicity complexes, which correlates with the tight binding of NNA to this isoform. Arginine and tetrahydrobiopterin promote loss of the flavin free radical EPR signal, while arginine analog inhibitors stabilize the radical; this suggests that the residual flavin radicals can serve as a source of reducing equivalents for slow turnover in the absence of endogenous reductant.

Authors+Show Affiliations

Salerno JC
Biology Department, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Martásek P
No affiliation info available
Williams RF
No affiliation info available
Masters BS
No affiliation info available

MeSH

AnimalsArginineCattleElectron Spin Resonance SpectroscopyEndothelium, VascularEnzyme InhibitorsNitric Oxide SynthaseNitroarginineProtein BindingSubstrate Specificityomega-N-Methylarginine

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9305973

Citation

Salerno, J C., et al. "Substrate and Substrate Analog Binding to Endothelial Nitric Oxide Synthase: Electron Paramagnetic Resonance as an Isoform-specific Probe of the Binding Mode of Substrate Analogs." Biochemistry, vol. 36, no. 39, 1997, pp. 11821-7.
Salerno JC, Martásek P, Williams RF, et al. Substrate and substrate analog binding to endothelial nitric oxide synthase: electron paramagnetic resonance as an isoform-specific probe of the binding mode of substrate analogs. Biochemistry. 1997;36(39):11821-7.
Salerno, J. C., Martásek, P., Williams, R. F., & Masters, B. S. (1997). Substrate and substrate analog binding to endothelial nitric oxide synthase: electron paramagnetic resonance as an isoform-specific probe of the binding mode of substrate analogs. Biochemistry, 36(39), 11821-7.
Salerno JC, et al. Substrate and Substrate Analog Binding to Endothelial Nitric Oxide Synthase: Electron Paramagnetic Resonance as an Isoform-specific Probe of the Binding Mode of Substrate Analogs. Biochemistry. 1997 Sep 30;36(39):11821-7. PubMed PMID: 9305973.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Substrate and substrate analog binding to endothelial nitric oxide synthase: electron paramagnetic resonance as an isoform-specific probe of the binding mode of substrate analogs. AU - Salerno,J C, AU - Martásek,P, AU - Williams,R F, AU - Masters,B S, PY - 1997/10/8/pubmed PY - 1997/10/8/medline PY - 1997/10/8/entrez SP - 11821 EP - 7 JF - Biochemistry JO - Biochemistry VL - 36 IS - 39 N2 - The binding of arginine analogs to endothelial nitric oxide synthase (eNOS, NOSIII) perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Using electron paramagnetic resonance as a probe of heme ligation geometry, four categories of high-spin complex could be distinguished. These are analogous to the four classes of high-spin complexes, stabilized individually by the binding of L-arginine, N-hydroxy-L-arginine (NHA), N-methyl-L-arginine (NMA), and N-nitro-L-arginine (NNA), which we have previously reported for the other two isoforms. Each of these species is five-coordinate and retains the axial thiolate ligand but each differs in its ligation geometry. N-Methyl-L-arginine is a relatively poor inhibitor of eNOS, and the NMA complex of eNOS differs from the N-methyl-L-arginine complexes of inducible nitric oxide synthase (iNOS, NOSII) and neuronal nitric oxide synthase (nNOS, NOSI) in that it is of lower rhombicity. We previously showed that inducible nitric oxide synthase, which binds NNA less tightly than eNOS and nNOS, could not form the lower rhombicity NNA complex characteristic of nNOS. Endothelial nitric oxide synthase readily forms such lower rhombicity complexes, which correlates with the tight binding of NNA to this isoform. Arginine and tetrahydrobiopterin promote loss of the flavin free radical EPR signal, while arginine analog inhibitors stabilize the radical; this suggests that the residual flavin radicals can serve as a source of reducing equivalents for slow turnover in the absence of endogenous reductant. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9305973/Substrate_and_substrate_analog_binding_to_endothelial_nitric_oxide_synthase:_electron_paramagnetic_resonance_as_an_isoform_specific_probe_of_the_binding_mode_of_substrate_analogs_ L2 - https://doi.org/10.1021/bi963003q DB - PRIME DP - Unbound Medicine ER -