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Transferencia de electrones acoplados a protones con puerta de histidina al sitio Cu_A de la óxido nitroso reductasa

Lin Zhang¹, Eckhard Bill², Peter M H Kroneck³

¹Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstraße 21, 79104 Freiburg im Breisgau, Germany.

Journal of the American Chemical Society

|December 30, 2020

Resumen

Este estudio revela cómo un residuo específico de histidina en la reductasa de óxido nitroso (N2OR) y sus enlaces de hidrógeno son cruciales para la función enzimática. La transferencia de electrones acoplados a protones en el sitio CuA es clave para convertir el gas de efecto invernadero N2O en N2.

Área de la Ciencia:

La bioquímica
Enzimología
Microbiología ambiental

Sus antecedentes:

La nitróxido reductasa (N2OR) es vital para la conversión del gas de efecto invernadero óxido nitroso (N2O) en nitrógeno (N2), completando la desnitrificación bacteriana.
La enzima presenta un sitio activo tetranuclear único (CuZ) y un sitio de entrada de electrones binuclear (CuA), también encontrado en otras enzimas como la citocromo c oxidasa.
En Pseudomonas stutzeri N2OR, un ligando de histidina en el sitio CuA exhibe cambios conformacionales en la unión con N2O.

Objetivo del estudio:

Investigar el papel de un residuo específico de histidina (His583) y su red de enlaces de hidrógeno en el sitio CuA de Pseudomonas stutzeri N2OR.
Para aclarar el mecanismo de transferencia de electrones en el sitio CuA durante la reducción de N2O.

Principales métodos:

Mutagénesis sistemática dirigida al sitio de His583 y residuos circundantes en un sistema de expresión funcional de PsN2OR en E. coli.
Caracterización espectroscópica y estructural de las variantes enzimáticas resultantes.
Evaluación de las capacidades de transferencia de electrones de las variantes a pH fisiológico.

Principales resultados:

Un único enlace de hidrógeno de Ser550 estabiliza la conformación no unida de His583.
La eliminación de este enlace de hidrógeno fuerza a His583 a una conformación unida, ligando a CuA.
Solo las variantes con cadenas laterales protonables (His, Asp, Glu) en la posición 583 mediaron la transferencia de electrones, lo que sugiere un mecanismo de transferencia de electrones acoplado a protones.

Conclusiones:

La flexibilidad conformacional de His583, regulada por la unión de hidrógeno, es esencial para la actividad de N2OR.
Los residuos protonables en el sitio CuA son críticos para mediar la transferencia de electrones en N2OR.
Los hallazgos apoyan un mecanismo de transferencia de electrones acoplado a protones en el sitio CuA de la reductasa de óxido nitroso.

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