title: X-ray absorption studies of Zn(2+)-binding sites in Escherichia coli transhydrogenase and its betaH91K mutant.
abstract: 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.
authors.affiliation: Dipartimento di Fisica, Università di Bologna, viale C. Berti Pichat 6/2, Bologna, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Veronesi
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Whitehead
authors.forename: Francesco
authors.initials: F
authors.lastname: Francia
authors.forename: Lisa
authors.initials: L
authors.lastname: Giachini
authors.forename: Federico
authors.initials: F
authors.lastname: Boscherini
authors.forename: Giovanni
authors.initials: G
authors.lastname: Venturoli
authors.forename: Nick P J
authors.initials: NP
authors.lastname: Cotton
authors.forename: J Baz
authors.initials: JB
authors.lastname: Jackson
mesh_terms: D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013052:Spectrometry, X-Ray Emission / Q000379:methods*; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D009250:NADP Transhydrogenases; D015032:Zinc; D003545:Cysteine