Determination of the midpoint potential of the FAD and FMN flavin cofactors and of the 3Fe-4S cluster of glutamate synthase.
Glutamate synthase is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of the L-glutamine amide group to C(2) of 2-oxoglutarate, forming two molecules of L-glutamate. The bacterial enzyme is an alphabeta protomer, which contains one FAD (on the beta subunit, approximately 50 kDa), one FMN (on the alpha subunit, approximately 150 kDa), and three different Fe-S clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters at an unknown location). To address the problem of the intramolecular electron pathway, we have measured the midpoint potential values of the flavin cofactors and of the 3Fe-4S cluster of glutamate synthase in the isolated alpha and beta subunits and in the alphabeta holoenzyme. No detectable amounts of flavin semiquinones were observed during reductive titrations of the enzyme, indicating that the midpoint potential value of each flavin(ox)/flavin(sq) couple is, in all cases, significantly more negative than that of the corresponding flavin(sq)/flavin(hq) couple. Association of the two subunits to form the alphabeta protomer does not alter significantly the midpoint potential value of the FMN cofactor and of the 3Fe-4S cluster (approximately -240 and -270 mV, respectively), but it makes that of FAD some 40 mV less negative (approximately -340 mV for the beta subunit and -300 mV for FAD bound to the holoenzyme). Binding of the nonreducible NADP(+) analogue, 3-aminopyridine adenine dinucleotide phosphate, made the measured midpoint potential value of the FAD cofactor approximately 30-40 mV less negative in the isolated beta subunit, but had no effect on the redox properties of the alphabeta holoenzyme. This result correlates with the formation of a stable charge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated beta subunit, but not in the alphabeta holoenzyme. Binding of L-methionine sulfone, a glutamine analogue, had no significant effect on the redox properties of the enzyme cofactors. On the contrary, 2-oxoglutarate made the measured midpoint potential value of the 3Fe-4S cluster approximately 20 mV more negative in the isolated alpha subunit, but up to 100 mV less negative in the alphabeta holoenzyme as compared to the values of the corresponding free enzyme forms. These findings are consistent with electron transfer from the entry site (FAD) to the exit site (FMN) through the 3Fe-4S center of the enzyme and the involvement of at least one of the two low-potential 4Fe-4S centers, which are present in the glutamate synthase holoenzyme, but not in the isolated subunits. Furthermore, the data demonstrate a specific role of 2-oxoglutarate in promoting electron transfer from FAD to the 3Fe-4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxoglutarate is indeed consistent with the recently determined three-dimensional structure of the glutamate synthase alpha subunit, in which several polypeptide stretches are suitably positioned to mediate communication between substrate binding sites and the enzyme redox centers (FMN and the 3Fe-4S cluster) to tightly control and coordinate the individual reaction steps [Binda, C., et al. (2000) Structure 8, 1299-1308].
Ravasio
S
S
Dipartimento di Fisiologia e Biochimica Generali, Universitá degli Studi di Milano, , Italy.
Curti
B
B
Vanoni
M A
MA
D015806:Azospirillum brasilense / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism*; D005970:Glutamate Synthase / Q000378:metabolism*; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D011199:Potentiometry; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D005415:Flavins; D020035:Holoenzymes; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D005970:Glutamate Synthase