Arginine conversion to nitroxide by tetrahydrobiopterin-free neuronal nitric-oxide synthase. Implications for mechanism.

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Adak S, Wang Q, Stuehr DJ

Arginine conversion to nitroxide by tetrahydrobiopterin-free neuronal nitric-oxide synthase. Implications for mechanism.

J Biol Chem. 2000 Oct 27;275(43):33554-61.

PubMed ID
10945985 [ View in PubMed
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Abstract

We studied catalysis by tetrahydrobiopterin (H4B)-free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation to N(omega)-hydroxy-l-Arg (NOHA) and citrulline in both NADPH- and H(2)O(2)-driven reactions. Citrulline formation was time- and enzyme concentration-dependent but was uncoupled relative to NADPH oxidation, and generated nitrite and nitrate without forming NO. Similar results were observed when NOHA served as substrate. Steady-state and stopped-flow spectroscopy with the H4B-free enzyme revealed that a ferrous heme-NO complex built up after initiating catalysis in both NADPH- and H(2)O(2)-driven reactions, consistent with formation of nitroxyl as an immediate product. This differed from the H4B-replete enzyme, which formed a ferric heme-NO complex as an immediate product that could then release NO. We make the following conclusions. 1) H4B is not essential for Arg oxidation by nNOS, although it helps couple NADPH oxidation to product formation in both steps of NO synthesis. Thus, the NADPH- or H(2)O(2)-driven reactions form common heme-oxy species that can react with substrate in the presence or absence of H4B. 2) The sole essential role of H4B is to enable nNOS to generate NO instead of nitroxyl. On this basis we propose a new unified model for heme-dependent oxygen activation and H4B function in both steps of NO synthesis.

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Drug Targets
DrugTargetKindOrganismPharmacological ActionActions
CitrullineNitric oxide synthase, brainProteinHumans
Unknown
Not AvailableDetails