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Redox Function of Tetrahydrobiopterin and Effect of <scp>l</scp>-Arginine on Oxygen Binding in Endothelial Nitric Oxide Synthase
- Source :
- Biochemistry. 43:13137-13148
- Publication Year :
- 2004
- Publisher :
- American Chemical Society (ACS), 2004.
-
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.
- Subjects :
- Free Radicals
Nitric Oxide Synthase Type III
Heme
Spodoptera
Arginine
Nitric Oxide
Photochemistry
Endothelial NOS
Ferric Compounds
Biochemistry
Medicinal chemistry
Redox
chemistry.chemical_compound
Enos
Dihydrobiopterin
Two-Hybrid System Techniques
Animals
Humans
Ferrous Compounds
biology
Reducing equivalent
biology.organism_classification
Biopterin
Recombinant Proteins
Protein Structure, Tertiary
Oxygen
Nitric oxide synthase
Kinetics
chemistry
biology.protein
Cattle
Nitric Oxide Synthase
Oxidation-Reduction
Oxygen binding
Protein Binding
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 43
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....8bfa5be6221553197137d60b4c6244ce