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Ajuste de Pi-interacción de las propiedades del sitio activo de las metaloproteínas

Sachiko Yanagisawa¹, Peter B Crowley, Susan J Firbank

¹Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

Journal of the American Chemical Society

|October 23, 2008

Resumen

La investigación de las interacciones pi en las metaloproteínas de cobre revela que modulan la protonación de su ligando, afectando la actividad redox. Estas interacciones estabilizan la pseudoazurina pero favorecen la disociación en la plastocyanin, lo que afecta a la regulación de la transferencia de electrones.

Área de la Ciencia:

La bioquímica es la bioquímica.
Química bioorgánica Química bioorgánica.
Ingeniería de proteínas Ingeniería de proteínas.

Sus antecedentes:

Las metaloproteínas redox que contienen cobre, como la pseudoazurina y la plastocyanin, son cruciales para la transferencia de electrones.
Los ligandos de histidina (His) juegan un papel clave en la actividad redox de estas metaloproteínas.
La protonación y la disociación de sus ligandos del centro de cobre pueden regular la transferencia de electrones.

Objetivo del estudio:

Para investigar la influencia de las interacciones pi del sitio activo en el comportamiento de sus ligandos en las metaloproteínas de cobre.
Para entender cómo los pi-contactos diseñados afectan a la pKa de sus ligandos y propiedades redox.
Explorar el papel de los residuos específicos en la modulación de estas interacciones y sus consecuencias funcionales.

Principales métodos:

Mutagénesis dirigida al sitio para introducir residuos de fenilalanina (Phe) o triptófano (Trp) creando pi-contactos.
Técnicas espectroscópicas y electroquímicas para analizar los estados de protonación de su ligando y los valores de pKa.
La cinética de las enzimas y las mediciones de autointercambio de electrones para evaluar los impactos funcionales.

Principales resultados:

Los contactos pi diseñados entre residuos aromáticos introducidos y sus ligandos se confirmaron en variantes de pseudoazurina y plastocyanin.
Las interacciones Pi tuvieron efectos opuestos en el ligando pKa de His: estabilizando la forma coordinada en pseudoazurina y favoreciendo la protonación/disociación en plastocyanin.
Las mutaciones influyeron significativamente en el potencial de reducción de la pseudoazurina y el autointercambio de electrones, con Met16Phe afectando a la asociación de nitrito reductasa.

Conclusiones:

Las interacciones pi del sitio activo son un factor significativo en la regulación de la protonación de su ligando y la actividad redox de las metaloproteínas de cobre.
Las diferencias sutiles en la geometría del contacto pi conducen a resultados funcionales distintos en diferentes familias de proteínas.
El estado de protonación del ligando His, influenciado por las interacciones de la segunda esfera de coordinación, actúa como un interruptor impulsado por [H+] para la transferencia de electrones.

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