X-ray absorption studies of Zn(2+)-binding sites in Escherichia coli transhydrogenase and its betaH91K mutant.

Transhydrogenase couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane. The binding of Zn(2+) 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 Zn(2+) bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which betaHis91 was replaced by Lys (betaH91K). 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 Zn(2+). The XAFS analysis has identified a Zn(2+)-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 Zn(2+) 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 betaH91K. Evidently, betaHis91 is not directly involved in Zn(2+) binding. The locus of betaHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.

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