Oxygen-Induced Radical Intermediates in the nNOS Oxygenase Domain Regulated by L-Arginine, Tetrahydrobiopterin, and Thiol

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• 作者：Vladimí ; r Berka ; Lee-Ho Wang ; Ah-Lim Tsai
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：January 8, 2008
• 年：2008
• 卷：47
• 期：1
• 页码：405 - 420
• 全文大小：294K
• 年卷期：v.47,no.1(January 8, 2008)
• ISSN：1520-4995

文摘

Fully coupled nitric oxide synthase (NOS) catalyzes formation of nitric oxide (NO), L-citrulline,NADP⁺, and water from L-arginine, NADPH, and oxygen. Uncoupled or partially coupled NOS catalyzesthe synthesis of reactive oxygen species such as superoxide, hydrogen peroxide, and peroxynitrite, dependingon the availability of cofactor tetrahydrobiopterin (BH₄) and L-arginine during catalysis. We identifiedthree distinct oxygen-induced radical intermediates in the ferrous endothelial NOS oxygenase domain(eNOS_ox) with or without BH₄ and/or L-arginine [Berka, V., Wu, G., Yeh, H. C., Palmer, G., and Tsai,A.-L. (2004) J. Biol. Chem. 279, 32243-32251]. The effects of BH₄ and L-arginine on the oxygen-induced radical intermediates in the isolated neuronal NOS oxygenase domain (nNOS_ox) have been similarlyinvestigated by single-turnover stopped-flow and rapid-freeze quench EPR kinetic measurements in thepresence or absence of dithiothreitol (DTT). Like for eNOS_ox, we found different radical intermediates inthe reaction of ferrous nNOS_ox with oxygen. (1) nNOS_ox (without BH₄ or L-Arg) produces superoxide inthe presence or absence of DTT. (2) nNOS_ox (with BH₄ and L-Arg) yields a typical BH₄ radical in amanner independent of DTT. (3) nNOS_ox (with BH₄ and without L-Arg) yields a new radical. WithoutDTT, EPR showed a mixture of superoxide and biopterin radicals. With DTT, a new ~75 G wide radicalEPR was observed, different from the radical formed by eNOS_ox. (4) The presence of only L-arginine innNOS_ox (without BH₄ but with L-Arg) caused conversion of ~70% of superoxide radical to a novel radical,explaining how L-arginine decreases the level of superoxide production in nNOS_ox (without BH₄ but withL-Arg). The regulatory role of L-arginine in nNOS is thus very different from that in eNOS where substratewas only to decrease the rate of formation of superoxide but not the total amount of radical. The role ofDTT is also different. DTT prevents oxidation of BH₄ in both isoforms, but in nNOS, DTT also inhibitsoxidation of two key cysteines in nNOS_ox to prevent the loss of substrate binding. This new role of thiolfound only for nNOS may be significant in neurodegenerative diseases.