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La eliminación reductiva de H2 activa la nitrogenasa para reducir el enlace triple NN: Caracterización del

Dmitriy Lukoyanov¹, Nimesh Khadka², Zhi-Yong Yang²

¹Departments of Chemistry and Molecular Biosciences, Northwestern University , Evanston, Illinois 60208, United States.

Journal of the American Chemical Society

|August 17, 2016

Resumen

La enzima nitrogenasa utiliza un mecanismo de eliminación reductora/adición oxidativa para convertir el gas nitrógeno (N2) en amoníaco (NH3). Se identificó un intermediario clave de Janus, confirmando este mecanismo y resolviendo enigmas de larga data en la investigación de la fijación de nitrógeno.

Área de la Ciencia:

La bioquímica
Química bioorgánica
Enzimología

Sus antecedentes:

La nitrogenasa es una enzima crucial que cataliza la reducción del nitrógeno atmosférico (N2) al amoníaco (NH3).
La comprensión del mecanismo de fijación del nitrógeno es esencial para las aplicaciones biológicas e industriales.
Estudios anteriores propusieron un mecanismo de eliminación reductiva/adición oxidativa (re/oa), pero la evidencia experimental fue limitada.

Objetivo del estudio:

Validar experimentalmente el mecanismo propuesto para la reducción de N2 catalizada por la nitrogenasa.
Identificar y caracterizar los intermediarios clave involucrados en el ciclo catalítico.
Aclarar el papel del intermediario Janus en la fijación del nitrógeno.

Principales métodos:

Mutagénesis dirigida al sitio de la proteína MoFe (α-70 ((Val→Ile) mutante).
Crioatrapamiento de los intermediarios enzimáticos durante la reducción de N2.
La resonancia paramagnética de electrones (EPR), ENDOR y la espectroscopia fotofísica.
Estudios de etiquetado de isótopos (buffer H2O/D2 y viceversa).

Principales resultados:

Identificación del intermedio Janus (E4(4H)) como un estado clave que almacena cuatro equivalentes reductores.
Demostrar que el intermediario de Janus está implicado en la rotación de la nitrogenasa de tipo salvaje (WT).
Confirmación de que los hidruros/deutéridos puentes en el intermedio Janus no se intercambian con el disolvente.
Evidencia de la reversibilidad cinética del equilibrio re/oa y de una reacción termoneutral.
Se identificó el nivel intermedio de reducción de diazeno (HNNH) (E4 ((2N2H)).

Conclusiones:

El mecanismo re/oa explica con precisión el ciclo catalítico de la nitrogenasa y resuelve las restricciones mecánicas clave.
El intermedio de Janus es fundamental para la reducción de N2 por la enzima de tipo salvaje.
La nitrogenasa cataliza eficazmente la difícil transformación del N2 en NH3 mediante el mecanismo de re/oa validado.

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