Video Experimental Relacionado
Updated: Jun 26, 2026
08:23
Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds
Published on: February 16, 2022
El segundo paso de la reacción de la sintasa de óxido nítrico: evidencia de que el peroxo férrico es el oxidante
Joshua J Woodward1, Michelle M Chang, Nathaniel I Martin
1Department of Chemistry, QB3 Institute, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720-3220, USA.
Journal of the American Chemical Society
|January 9, 2009
Resumen
Este estudio investiga el mecanismo de la óxido nítrico sintasa (NOS) mediante el examen de sus oxidantes enlazados con el hemo. Los hallazgos sugieren que un intermediario férrico-peróxido forma citrulina, mientras que un intermediario Fe ((IV) O (((por ((*+)) de alta valencia produce productos secundarios.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Biología Química Biología química.
Sus antecedentes:
- La sintasa de óxido nítrico (NOS) es una enzima P450 que cataliza la oxidación de la L-arginina a citrulina y NO a través de la N(G) -hidroxi-L-arginina (NHA).
- Todavía no está clara la naturaleza exacta del oxidante unido al hemo (ferrico-peroxo vs. Fe(IV) O((por(*+))) en la oxidación del NHA.
- Estudios previos indicaron la participación de un intermediario férrico-peróxido en la formación de citrulina y N ((delta) -cianornitina (CN-orn) como un producto secundario.
Objetivo del estudio:
- Para dilucidar el papel de diferentes oxidantes unidos al hemo en la oxidación de NHA catalizada por NOS.
- Para investigar la influencia del pH, la sustitución del hemo, la mutagénesis del sitio activo y los análogos del sustrato en la distribución del producto.
- Para sondear la descomposición catalizada por proteínas del peróxido al intermedio Fe(IV) O(por(*+)).
Principales métodos:
- Se utilizó una reacción modelo que involucra la oxidación impulsada por H(2) O(2) de NHA por NOS.
- Condiciones de reacción variadas: pH (6.5, 7.5, 8.5), cofactor hemo (Fe-MPIX, Mn) y mutación del sitio activo (V346A).
- Se empleó un análogo de sustrato fluorado (4,4-difluoro-N(G) -hidroxi-L-arginina) y el ensayo ABTS para estudiar la actividad de la peroxidasa.
Principales resultados:
- La formación de citrulina aumentó con el pH (26-51%), mientras que la actividad de la peroxidasa disminuyó.
- La mutación V346A disminuyó la producción de citrulina (5,2%) pero mejoró la escisión de peróxido.
- La sustitución de hemo (Fe-MPIX, Mn) y el análogo del sustrato alteraron las proporciones del producto, con Mn-hemo dando el 64,7% de citrulina y el análogo dando el 68,6% de difluorocitrulina.
- La descomposición de peróxido se correlacionó negativamente con la formación de citrulina, apoyando el Fe ((IV) O (((por ((*+)) en la producción de CN-orn y el peróxido férrico en la formación de citrulina.
Conclusiones:
- El estudio proporciona información sobre las características químicas de NOS que rigen el procesamiento intermedio de férrico-peróxido a Fe ((IV) O (((por ((*+)).
- Los resultados apoyan el papel del intermediario férrico-peróxido en la oxidación del NHA a la citrulina.
- El intermedio de alta valencia Fe ((IV) O (((por ((*+)) está implicado en la formación del producto secundario CN-orn.
Más Videos Relacionados
08:32Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
13:21Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps
Published on: August 18, 2012
Videos de Conceptos Relacionados
03:08Rate-Determining Steps
Relating Reaction Mechanisms
In a multistep reaction mechanism, one of the elementary steps progresses significantly slower than the others. This slowest step is called the rate-limiting step (or rate-determining step). A reaction cannot proceed faster than its slowest step, and hence, the rate-determining step limits the overall reaction rate.
The concept of rate-determining step can be understood from the analogy of a 4-lane freeway with a short-stretch of traffic-bottleneck caused due to...
In a multistep reaction mechanism, one of the elementary steps progresses significantly slower than the others. This slowest step is called the rate-limiting step (or rate-determining step). A reaction cannot proceed faster than its slowest step, and hence, the rate-determining step limits the overall reaction rate.
The concept of rate-determining step can be understood from the analogy of a 4-lane freeway with a short-stretch of traffic-bottleneck caused due to...
01:17Oxidation of Phenols to Quinones
In the presence of oxidizing agents, phenols are oxidized to quinones. Quinones can be easily reduced back to phenols using mild reducing agents. The electron-donating hydroxyl group enhances the reactivity of the aromatic ring, enabling oxidation of the ring even in the absence of an α hydrogen.
o-hydroxy phenols are oxidized to o-quinones and p-hydroxy phenols to p-quinones. Such redox reactions involve the transfer of two electrons and two protons. The reversible redox property is crucial in...
o-hydroxy phenols are oxidized to o-quinones and p-hydroxy phenols to p-quinones. Such redox reactions involve the transfer of two electrons and two protons. The reversible redox property is crucial in...
02:31Multi-Step Reactions
Chemical reactions often occur in a stepwise fashion involving two or more distinct reactions taking place in a sequence. A balanced equation indicates the reacting species and the product species, but it reveals no details about how the reaction occurs at the molecular level. The reaction mechanism (or reaction path) provides details regarding the precise, step-by-step process by which a reaction occurs. Each of the steps in a reaction mechanism is called an elementary reaction. These...
01:202° Amines to N-Nitrosamines: Reaction with NaNO2
Secondary amines react with nitrous acid to form N-nitrosamines, as depicted in Figure 1. Nitrous acid, a weak and unstable acid, is formed in situ from an aqueous solution of sodium nitrite and strong acids, such as hydrochloric acid or sulfuric acid, in cold conditions. In the presence of an acid, the nitrous acid gets protonated. The subsequent loss of water results in the formation of the electrophile known as nitrosonium ion.
01:28Nitric Oxide Signaling Pathway
Nitric oxide (NO), an inorganic gas, acts as a potent second messenger in most animal and plant tissues. NO diffuses out of the cells that produce it and enters the neighboring cells to generate a downstream response. NO synthase (NOS) catalyzes NO production by the deamination of the amino acid arginine. There are three isoforms of NOS. Endothelial cells have endothelial NOS (eNOS), nerve and muscle cells have neuronal NOS (nNOS), and macrophages produce inducible NOS (iNOS) upon exposure to...
01:16Necrosis
Necrosis is considered as an “accidental” or unexpected form of cell death that ends in cell lysis. The first noticeable mention of “necrosis” was in 1859 when Rudolf Virchow used this term to describe advanced tissue breakdown in his compilation titled “Cell Pathology”.
Morphological Manifestations of Necrosis
Necrotic cells show different types of morphological appearance depending on the type of tissue and infection. In coagulative necrosis, cells become anucleated and die, but their...
Morphological Manifestations of Necrosis
Necrotic cells show different types of morphological appearance depending on the type of tissue and infection. In coagulative necrosis, cells become anucleated and die, but their...