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Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase.
Biochemistry. 2004 Oct 19; 43(41):13137-48.B

Abstract

Tetrahydrobiopterin (BH(4)), not dihydrobiopterin or biopterin, is a critical element required for NO formation by nitric oxide synthase (NOS). To elucidate how BH(4) affects eNOS activity, we have investigated BH(4) redox functions in the endothelial NOS (eNOS). Redox-state changes of BH(4) in eNOS were examined by chemical quench/HPLC analysis during the autoinactivation of eNOS using oxyhemoglobin oxidation assay for NO formation at room temperature. Loss of NO formation activity linearly correlated with BH(4) oxidation, and was recovered by overnight incubation with fresh BH(4). Thus, thiol reagents commonly added to NOS enzyme preparations, such as dithiothreitol and beta-mercaptoethanol, probably preserve enzyme activity by preventing BH(4) oxidation. It has been shown that conversion of L-arginine to N-hydroxy-L-arginine in the first step of NOS catalysis requires two reducing equivalents. The first electron that reduces ferric to the ferrous heme is derived from flavin oxidation. The issue of whether BH(4) supplies the second reducing equivalent in the monooxygenation of eNOS was investigated by rapid-scan stopped-flow and rapid-freeze-quench EPR kinetic measurements. In the presence of L-arginine, oxygen binding kinetics to ferrous eNOS or to the ferrous eNOS oxygenase domain (eNOS(ox)) followed a sequential mechanism: Fe(II) <--> Fe(II)O(2) --> Fe(III) + O(2)(-). Without L-arginine, little accumulation of the Fe(II)O(2) intermediate occurred and essentially a direct optical transition from the Fe(II) form to the Fe(III) form was observed. Stabilization of the Fe(II)O(2) intermediate by L-arginine has been established convincingly. On the other hand, BH(4) did not have significant effects on the oxygen binding and decay of the oxyferrous intermediate of the eNOS or eNOS oxygenase domain. Rapid-freeze-quench EPR kinetic measurements in the presence of L-arginine showed a direct correlation between BH(4) radical formation and decay of the Fe(II)O(2) intermediate, indicating that BH(4) indeed supplies the second electron for L-arginine monooxygenation in eNOS.

Authors+Show Affiliations

Berka V
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
Yeh HC
No affiliation info available
Gao D
No affiliation info available
Kiran F
No affiliation info available
Tsai AL
No affiliation info available

MeSH

AnimalsArginineBiopterinCattleFerric CompoundsFerrous CompoundsFree RadicalsHemeHumansKineticsNitric OxideNitric Oxide SynthaseNitric Oxide Synthase Type IIIOxidation-ReductionOxygenProtein BindingProtein Structure, TertiaryRecombinant ProteinsSpodopteraTwo-Hybrid System Techniques

Pub Type(s)

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

Language

eng

PubMed ID

15476407

Citation

Berka, Vladimir, et al. "Redox Function of Tetrahydrobiopterin and Effect of L-arginine On Oxygen Binding in Endothelial Nitric Oxide Synthase." Biochemistry, vol. 43, no. 41, 2004, pp. 13137-48.
Berka V, Yeh HC, Gao D, et al. Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase. Biochemistry. 2004;43(41):13137-48.
Berka, V., Yeh, H. C., Gao, D., Kiran, F., & Tsai, A. L. (2004). Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase. Biochemistry, 43(41), 13137-48.
Berka V, et al. Redox Function of Tetrahydrobiopterin and Effect of L-arginine On Oxygen Binding in Endothelial Nitric Oxide Synthase. Biochemistry. 2004 Oct 19;43(41):13137-48. PubMed PMID: 15476407.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase. AU - Berka,Vladimir, AU - Yeh,Hui-Chun, AU - Gao,De, AU - Kiran,Farheen, AU - Tsai,Ah-Lim, PY - 2004/10/13/pubmed PY - 2004/11/13/medline PY - 2004/10/13/entrez SP - 13137 EP - 48 JF - Biochemistry JO - Biochemistry VL - 43 IS - 41 N2 - Tetrahydrobiopterin (BH(4)), not dihydrobiopterin or biopterin, is a critical element required for NO formation by nitric oxide synthase (NOS). To elucidate how BH(4) affects eNOS activity, we have investigated BH(4) redox functions in the endothelial NOS (eNOS). Redox-state changes of BH(4) in eNOS were examined by chemical quench/HPLC analysis during the autoinactivation of eNOS using oxyhemoglobin oxidation assay for NO formation at room temperature. Loss of NO formation activity linearly correlated with BH(4) oxidation, and was recovered by overnight incubation with fresh BH(4). Thus, thiol reagents commonly added to NOS enzyme preparations, such as dithiothreitol and beta-mercaptoethanol, probably preserve enzyme activity by preventing BH(4) oxidation. It has been shown that conversion of L-arginine to N-hydroxy-L-arginine in the first step of NOS catalysis requires two reducing equivalents. The first electron that reduces ferric to the ferrous heme is derived from flavin oxidation. The issue of whether BH(4) supplies the second reducing equivalent in the monooxygenation of eNOS was investigated by rapid-scan stopped-flow and rapid-freeze-quench EPR kinetic measurements. In the presence of L-arginine, oxygen binding kinetics to ferrous eNOS or to the ferrous eNOS oxygenase domain (eNOS(ox)) followed a sequential mechanism: Fe(II) <--> Fe(II)O(2) --> Fe(III) + O(2)(-). Without L-arginine, little accumulation of the Fe(II)O(2) intermediate occurred and essentially a direct optical transition from the Fe(II) form to the Fe(III) form was observed. Stabilization of the Fe(II)O(2) intermediate by L-arginine has been established convincingly. On the other hand, BH(4) did not have significant effects on the oxygen binding and decay of the oxyferrous intermediate of the eNOS or eNOS oxygenase domain. Rapid-freeze-quench EPR kinetic measurements in the presence of L-arginine showed a direct correlation between BH(4) radical formation and decay of the Fe(II)O(2) intermediate, indicating that BH(4) indeed supplies the second electron for L-arginine monooxygenation in eNOS. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15476407/Redox_function_of_tetrahydrobiopterin_and_effect_of_L_arginine_on_oxygen_binding_in_endothelial_nitric_oxide_synthase_ DB - PRIME DP - Unbound Medicine ER -