title: Electrostatic control of the isoalloxazine environment in the two-electron reduced states of yeast glutathione reductase.
abstract: The resonance Raman spectra of the oxidized and two-electron reduced forms of yeast glutathione reductase are reported. The spectra of the oxidized enzyme indicate a low electron density for the isoalloxazine ring. As far as the two-electron reduced species are concerned, the spectral comparison of the NADPH-reduced enzyme with the glutathione- or dithiothreitol-reduced enzyme shows significant frequency differences for the flavin bands II, III, and VII. The shift of band VII was correlated with a change in steric or electronic interaction of the hydroxyl group of a conserved Tyr with the N(10)-C(10a) portion of the isoalloxazine ring. Upward shifts of bands II and III observed for the glutathione- or dithiothreitol-reduced enzyme indicate both a slight change in isoalloxazine conformation and a hydrogen bond strengthening at the N(1) and/or N(5) site(s). The formation of a mixed disulfide intermediate tends to slightly decrease the frequency of bands II, III, X, XI, and XIV. To account for the different spectral features observed for the NADPH- and glutathione-reduced species, several possibilities have been examined. In particular, we propose a hydrogen bonding modulation at the N(5) site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N(5)(flavin)-protein interaction in the two-electron reduced forms of glutathione reductase are discussed in relation to a plausible mechanism of the regulation of the enzyme activity via a variable redox potential of FAD.
authors.affiliation: Département de Biologie Joliot-Curie, Section de Biophysique des Fonctions Membranaires, Commissariat à l'Energie (CEA), F-91191 Gif-sur-Yvette Cedex, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Picaud
authors.forename: Alain
authors.initials: A
authors.lastname: Desbois
mesh_terms: D001665:Binding Sites; D005182:Flavin-Adenine Dinucleotide; D005415:Flavins; D005980:Glutathione Reductase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013059:Spectrum Analysis, Raman; D055672:Static Electricity
chemical_list: D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; C008173:isoalloxazine; D005980:Glutathione Reductase