title: Inhibition of proton-transfer steps in transhydrogenase by transition metal ions.
abstract: Transhydrogenase couples proton translocation across a bacterial or mitochondrial membrane to the redox reaction between NAD(H) and NADP(H). Purified intact transhydrogenase from Escherichia coli was prepared, and its His tag removed. The forward and reverse transhydrogenation reactions catalysed by the enzyme were inhibited by certain metal ions but a "cyclic reaction" was stimulated. Of metal ions tested they were effective in the order Pb(2+)>Cu(2+)>Zn(2+)=Cd(2+)>Ni(2+)>Co(2+). The results suggest that the metal ions affect transhydrogenase by binding to a site in the proton-transfer pathway. Attenuated total-reflectance Fourier-transform infrared difference spectroscopy indicated the involvement of His and Asp/Glu residues in the Zn(2+)-binding site(s). A mutant in which betaHis91 in the membrane-spanning domain of transhydrogenase was replaced by Lys had enzyme activities resembling those of wild-type enzyme treated with Zn(2+). Effects of the metal ion on the mutant were much diminished but still evident. Signals in Zn(2+)-induced FTIR difference spectra of the betaHis91Lys mutant were also attributable to changes in His and Asp/Glu residues but were much smaller than those in wild-type spectra. The results support the view that betaHis91 and nearby Asp or Glu residues participate in the proton-transfer pathway of transhydrogenase.
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Whitehead
authors.forename: Masayo
authors.initials: M
authors.lastname: Iwaki
authors.forename: Nick P J
authors.initials: NP
authors.lastname: Cotton
authors.forename: Peter R
authors.initials: PR
authors.lastname: Rich
authors.forename: J Baz
authors.initials: JB
authors.lastname: Jackson
mesh_terms: D055162:Biocatalysis / Q000187:drug effects; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006865:Hydrogenation / Q000187:drug effects; D007477:Ions; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D015032:Zinc / Q000378:metabolism*
chemical_list: D003902:Detergents; D007477:Ions; D008081:Liposomes; D050505:Mutant Proteins; D009711:Nucleotides; D011522:Protons; D009250:NADP Transhydrogenases; D015032:Zinc