title: Cross-linking and N-(1-pyrenyl)maleimide labeling of cysteine mutants of proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli.
abstract: The pyridine nucleotide transhydrogenase of Escherichiacoli is a proton pump composed of two subunits (alpha and beta) organized as an alpha2beta2 tetramer. The enzyme contains seven cysteine residues, five in the alpha-subunit and two in the beta-subunit. The reaction of these residues with the cross-linking agent cupric 1, 10-phenanthrolinate and with the fluorescent thiol reagent N-(1-pyrenyl)maleimide was investigated in mutants in which one or more of these cysteine residues had been mutated to serine or threonine residues. Mutation of alphaCys395 and alphaCys397 prevented disulfide bond formation to give the cross-linked alpha2 dimer. We concluded that the two alpha-subunits of the holoenzyme interface in the region of these two cysteine residues. Pyrenylmaleimide reacted with detergent-washed cytoplasmic membrane vesicles containing high levels of transhydrogenase protein to show characteristic fluorescence emission bands at 378-379, 397-398, and 419-420 nm. At higher ratios of pyrenylmaleimide:transhydrogenase (>5:1) and longer times of reaction, an eximer band at 470 nm was formed. This was attributed to interaction between noncovalently bound molecules of pyrenylmaleimide. The cysteine residues of the beta-subunit (betaCys147 and betaCys260) were covalently modified by pyrenylmaleimide. betaCys147 reacted more strongly than betaCys260 with the fluorophore, and the pyrene derivative of betaCys147 was more accessible to quenching by 5-doxylstearate, suggesting a proximity to the surface of the membrane. Covalent modification of betaCys260 resulted in inhibition of enzyme activity. The inhibition was attributed to the introduction of the bulky pyrene group into the enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: E G
authors.initials: EG
authors.lastname: Sedgwick
authors.forename: J
authors.initials: J
authors.lastname: Meuller
authors.forename: C
authors.initials: C
authors.lastname: Hou
authors.forename: J
authors.initials: J
authors.lastname: Rydström
authors.forename: P D
authors.initials: PD
authors.lastname: Bragg
mesh_terms: D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D008301:Maleimides / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics; D017494:Proton Pumps / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D003432:Cross-Linking Reagents; D008301:Maleimides; D017494:Proton Pumps; C003944:N-(3-pyrene)maleimide; D009250:NADP Transhydrogenases; D003545:Cysteine