Is the Bound Substrate in Nitric Oxide Synthase Protonated or Neutral and What Is the Active Oxidant that Performs Substrate Hydroxylation?
We present here results of a series of density functional theory (DFT) studies on enzyme active site models of nitric oxide synthase (NOS) and address the key steps in the catalytic cycle whereby the substrate (L-arginine) is hydroxylated to N-hydroxo-arginine. It has been proposed that the mechanis...
| Publicado en: | Journal of the American Chemical Society Vol. 130; no. 39; pp. 12961 - 12975 |
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| Autores principales: | , |
| Formato: | Bibliografía |
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American Chemical Society
10/1/2008
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=hlh&AN=34624300&site=ehost-live header: @attributes: shortDbName: hlh uiTerm: 34624300 longDbName: Humanities International Complete uiTag: AN controlInfo: bkinfo: jinfo: jid: 00027863 ACS jtl: Journal of the American Chemical Society issn: 00027863 maglogo: N pubinfo: dt: 10/1/2008 vid: 130 iid: 39 pid: 997 pub: American Chemical Society artinfo: ui: 34624300 10.1021/ja8010995 ppf: 12961 ppct: 14 formats: tig: atl: Is the Bound Substrate in Nitric Oxide Synthase Protonated or Neutral and What Is the Active Oxidant that Performs Substrate Hydroxylation? aug: au: de Visser, Sam P. Lee Siew Tan affil: Manchester Interdisciplinary Biocenter and School of Chemical Engineering and Analytical Science, University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom su: Density functionals Enzymes Nitric oxide Reactive oxygen species Catalysis Protons sug: subj: Density functionals Enzymes Nitric oxide Reactive oxygen species Catalysis Protons ab: We present here results of a series of density functional theory (DFT) studies on enzyme active site models of nitric oxide synthase (NOS) and address the key steps in the catalytic cycle whereby the substrate (L-arginine) is hydroxylated to N-hydroxo-arginine. It has been proposed that the mechanism follows a cytochrome P450-type catalytic cycle; however, our calculations find an alternative low energy pathway whereby the bound L-arginine substrate has two important functions in the catalytic cycle, namely first as a proton donor and later as the substrate in the reaction mechanism. Thus, the DFT studies show that the oxo-iron active species (compound I) cannot abstract a proton and neither a hydrogen atom from protonated L-arginine due to the strength of the N-H bonds of the substrate. However, the hydroxylation of neutral arginine by compound I and its one electron reduced form (compound II) requires much lower barriers and is highly exothermic. Detailed analysis of proton transfer mechanisms shows that the basicity of the dioxo dianion and the hydroperoxo-iron (compound 0) intermediates in the catalytic cycle are larger than that of arginine, which makes it likely that protonated arginine donates one of the two protons needed during the first catalytic cycle of NOS. Therefore, DFT predicts that in NOS enzymes arginine binds to the active site in its protonated form, but is deprotonated during the oxygen activation process in the catalytic cycle by either the dioxo dianion species or compound 0. As a result of the low ionization potential of neutral arginine, the actual hydroxylation reaction starts with an initial electron transfer from the substrate to compound Ito create compound II followed by a concerted hydrogen abstraction/radical rebound from the substrate. These studies indicate that compound II is the actual oxidant in NOS enzymes that performs the hydroxylation reaction of arginine, which is in sharp contrast with the cytochromes P450 where compound II was shown to be a sluggish oxidant. This is the first example of an enzyme where compound II is able to participate in the reaction mechanism. Moreover, arginine hydroxylation by NOS enzymes is catalyzed in a significantly different way from the cytochromes P450 although the active sites of the two enzyme classes are very similar in structure. Detailed studies of environmental effects on the reaction mechanism show that environmental perturbations as appear in the protein have little effect and do not change the energies of the reaction. Finally, a valence bond curve crossing model has been set up to explain the obtained reaction mechanisms for the hydrogen abstraction processes in P450 and NOS enzymes. pubtype: Academic Journal doctype: Bibliography src: R language: English refInfo: copyright: @attributes: flag: Y dt: @attributes: year: 2008 holdings: @attributes: islocal: N |
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