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La introducción de un centro de hierro no hemo 2-His-1-Glu en la mioglobina confiere la actividad de la óxido nítrico
Ying-Wu Lin1, Natasha Yeung, Yi-Gui Gao
1Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Journal of the American Chemical Society
|July 1, 2010
Resumen
Los investigadores diseñaron un nuevo centro metálico 2-His-1-Glu en la mioglobina del cachalote, creando un nuevo modelo de óxido nítrico reductasa (NOR). Esta proteína de ingeniería demostró una actividad NOR significativa con cobre o hierro, ofreciendo información sobre la función de la enzima.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Ingeniería de enzimas Ingeniería de enzimas Ingeniería de enzimas
- Biología Estructural Biología estructural.
Sus antecedentes:
- Las enzimas de hierro no hemo utilizan un centro metálico 2-His-1-Glu conservado.
- La mioglobina del cachalote (swMb) es una proteína bien caracterizada, susceptible a la ingeniería.
Objetivo del estudio:
- Para diseñar un centro metálico 2-His-1-Glu en el swMb.
- Para investigar la actividad de la óxido nítrico reductasa (NOR) de la proteína modificada con diferentes iones metálicos.
- Para aclarar el mecanismo de la actividad de NOR a través de estudios estructurales y espectroscópicos.
Principales métodos:
- La mutagénesis dirigida al sitio se utilizó para diseñar el centro metálico 2-His-1-Glu en swMb (Fe(B) Mb(-His)).
- La cristalografía de rayos X de alta resolución determinó la estructura de Cu (II) -CN (II) -Fe (B) -Mb (His).
- La espectroscopia de resonancia paramagnética de electrones (EPR) analizó los intermediarios de reacción durante la reducción del óxido nítrico.
Principales resultados:
- Se diseñó con éxito un centro metálico funcional 2-His-1-Glu en swMb.
- Tanto Cu (I) -Fe (B) -Mb (His) como Fe (II) -Fe (B) -Mb (His) exhibieron actividad de la reductasa del óxido nítrico (NOR), con el cobre mostrando una mayor actividad.
- Los datos estructurales y EPR revelaron distintos intermediarios para las enzimas ligadas a Cu y Fe, destacando el papel del ligando metálico y el metal ligado.
Conclusiones:
- El Fe(B) Mb(-His) modificado sirve como un nuevo modelo de proteína para NOR.
- Tanto el ligando metálico diseñado (Glu29) como el ion metálico unido (Cu o Fe) son críticos para la actividad de NOR.
- Este trabajo proporciona información sobre la recién identificada familia de enzimas gNOR.
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