Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin.
Endothelial nitric-oxide synthase (eNOS) plays important roles in vascular physiology and homeostasis. Whether eNOS catalyzes nitric oxide biosynthesis or the synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite is dictated by the bioavailability of tetrahydrobiopterin (BH(4)) and L-arginine during eNOS catalysis. The effect of BH(4) and L-arginine on oxygen-induced radical intermediates has been investigated by single turnover rapid-freeze quench and EPR spectroscopy using the isolated eNOS oxygenase domain (eNOS(ox)). Three distinct radical intermediates corresponding to >50% of the heme were observed during the reaction between ferrous eNOS(ox) and oxygen. BH(4)-free eNOS(ox) produced the superoxide radical very efficiently in the absence of L-arginine. L-Arginine decreased the formation rate of superoxide by an order of magnitude but not its final level or EPR line shape. For BH(4)-containing eNOS(ox), only a stoichiometric amount of BH(4) radical was produced in the presence of L-arginine, but in its absence a new radical was obtained. This new radical could be either a peroxyl radical of BH(4) or an amino acid radical was in the vicinity of the heme. Formation of this new radical is very rapid, >150 s(-1), and it was subsequently converted to a BH(4) radical. The trapping of the superoxide radical by cytochrome c in the reaction of BH(4)(-) eNOS(ox) exhibited a limiting rate of approximately 15 s(-1), the time for the superoxide radical to leave the heme pocket and reach the protein surface; this reveals a general problem of the regular spin-trapping method in determining radical formation kinetics. Cytochrome c failed to trap the new radical species. Together with other EPR characteristics, our data strongly support the conclusion that this new radical is not a superoxide radical or a mixture of superoxide and biopterin radicals. Our study points out distinct roles of BH(4) and L-arginine in regulating eNOS radical intermediates. BH(4) prevented superoxide formation by chemical conversions of the Fe(II)O(2) intermediate, and l-arginine delayed superoxide formation by electronic interaction with the heme-bound oxygen.
Berka
Vladimir
V
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, 77030, USA.
Wu
Gang
G
Yeh
Hui-Chun
HC
Palmer
Graham
G
Tsai
Ah-lim
AL
D000596:Amino Acids / Q000737:chemistry; D001120:Arginine / Q000737:chemistry*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry*; D002384:Catalysis; D045304:Cytochromes c / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006418:Heme / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III; D016011:Normal Distribution; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013481:Superoxides / Q000737:chemistry; D013997:Time Factors
D000596:Amino Acids; D005609:Free Radicals; D011506:Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013481:Superoxides; D001708:Biopterins; D006418:Heme; D045304:Cytochromes c; D001120:Arginine; D010105:Oxygenases; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin; D010100:Oxygen