Protein Dynamics in the Reductive Activation of a B12-Containing Enzyme
| Autor: | Sebastian Goetzl, Holger Dobbek, Sandra E. Hennig, Robert Bittl, Jae-Hun Jeoung, Christian Teutloff, Tobias Werther |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Iron-Sulfur Proteins Models Molecular Conformational change crystal structure conformational transitions Stereochemistry Protein Conformation Coenzymes Enzyme Activators Firmicutes Crystallography X-Ray Biochemistry 03 medical and health sciences Electron transfer chemistry.chemical_compound Protein structure Corrinoid Bacterial Proteins Multienzyme Complexes Protein Interaction Domains and Motifs Databases Protein Chemistry Protein dynamics kinetic parameters 500 Naturwissenschaften und Mathematik::530 Physik::530 Physik monomers Aldehyde Oxidoreductases proteins Recombinant Proteins Kinetics Protein Subunits Vitamin B 12 030104 developmental biology Förster resonance energy transfer Catalytic cycle Amino Acid Substitution Solubility Mutation peptides Mutagenesis Site-Directed Anaerobic bacteria Protein Multimerization Dimerization Oxidation-Reduction |
| Zdroj: | Biochemistry. 56(41) |
| ISSN: | 1520-4995 |
| Popis: | 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 RACo:CoFeSP complex, consistent with the recent crystal structures. By analyzing the transient kinetics of formation and dissociation of the RACo:CoFeSP 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. |
| Databáze: | OpenAIRE |
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