Transhydrogenase couples hydride transfer between NADH and NADP+ to proton translocation across a membrane. The binding of Zn2+ to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn2+ bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which βHis91 was replaced by Lys (βH91K). This well-conserved His residue, located in the membrane spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn2+. The XAFS analysis has identified a Zn2+-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn2+ inhibits proton translocation by competing with H+ binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and βH91K. Evidently, βHis91 is not directly involved in Zn2+ binding. The locus of βHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.

G. Venturoli, F. Boscherini, J. B. Jackson, F. Francia, G. Veronesi (2009). XAFS study of the Zn binding site in native and genetically modified transhydrogenase. SC-2540..

XAFS study of the Zn binding site in native and genetically modified transhydrogenase. SC-2540.

VENTUROLI, GIOVANNI;BOSCHERINI, FEDERICO;FRANCIA, FRANCESCO;VERONESI, GIULIA
2009

Abstract

Transhydrogenase couples hydride transfer between NADH and NADP+ to proton translocation across a membrane. The binding of Zn2+ to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn2+ bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which βHis91 was replaced by Lys (βH91K). This well-conserved His residue, located in the membrane spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn2+. The XAFS analysis has identified a Zn2+-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn2+ inhibits proton translocation by competing with H+ binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and βH91K. Evidently, βHis91 is not directly involved in Zn2+ binding. The locus of βHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.
2009
2009
G. Venturoli, F. Boscherini, J. B. Jackson, F. Francia, G. Veronesi (2009). XAFS study of the Zn binding site in native and genetically modified transhydrogenase. SC-2540..
G. Venturoli; F. Boscherini; J. B. Jackson; F. Francia; G. Veronesi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/152011
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