title: Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies.
abstract: Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.
authors.affiliation: Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
authors.forename: M A
authors.initials: MA
authors.lastname: Vanoni
authors.forename: D E
authors.initials: DE
authors.lastname: Edmondson
authors.forename: G
authors.initials: G
authors.lastname: Zanetti
authors.forename: B
authors.initials: B
authors.lastname: Curti
mesh_terms: D015806:Azospirillum brasilense / Q000201:enzymology*; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005415:Flavins / Q000737:chemistry*; D005970:Glutamate Synthase / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008027:Light; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D013447:Sulfites / Q000737:chemistry; D013455:Sulfur / Q000737:chemistry; D013696:Temperature
chemical_list: D005415:Flavins; D007506:Iron-Sulfur Proteins; D013447:Sulfites; D009249:NADP; D013455:Sulfur; D007501:Iron; D005970:Glutamate Synthase