Protein Dynamics in the Reductive Activation of a B12-Containing Enzyme.
Biochemistry
; 56(41): 5496-5502, 2017 10 17.
Article
em En
| MEDLINE
| ID: mdl-28949132
ABSTRACT
B12-dependent proteins are involved in methyl transfer reactions ranging from the biosynthesis of methionine in humans to the formation of acetyl-CoA in anaerobic bacteria. During their catalytic cycle, they undergo large conformational changes to interact with various proteins. Recently, the crystal structure of the B12-containing corrinoid iron-sulfur protein (CoFeSP) in complex with its reductive activator (RACo) was determined, providing a first glimpse of how energy is transduced in the ATP-dependent reductive activation of corrinoid-containing methyltransferases. The thermodynamically uphill electron transfer from RACo to CoFeSP is accompanied by large movements of the cofactor-binding domains of CoFeSP. To refine the structure-based mechanism, we analyzed the conformational change of the B12-binding domain of CoFeSP by pulsed electron-electron double resonance and Förster resonance energy transfer spectroscopy. We show that the site-specific labels on the flexible B12-binding domain and the small subunit of CoFeSP move within 11 Å in the RACoCoFeSP complex, consistent with the recent crystal structures. By analyzing the transient kinetics of formation and dissociation of the RACoCoFeSP complex, we determined values of 0.75 µM-1 s-1 and 0.33 s-1 for rate constants kon and koff, respectively. Our results indicate that the large movement observed in crystals also occurs in solution and that neither the formation of the protein encounter complex nor the large movement of the B12-binding domain is rate-limiting for the ATP-dependent reductive activation of CoFeSP by RACo.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Vitamina B 12
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Modelos Moleculares
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Coenzimas
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Ativadores de Enzimas
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Firmicutes
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Proteínas Ferro-Enxofre
Idioma:
En
Revista:
Biochemistry
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Alemanha