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-oxoglutarat e, forming two molecules of L-glutamate. The bacterial enzyme is an alpha b eta protomer, which contains one FAD ton the beta subunit, similar to 50 kD a), one FMN ton the alpha subunit, similar to 150 kDa), and three different Fe-S clusters tone 3Fe-4S center on the alpha subunit and two 4Fe-4S clust ers at an unknown location). To address the problem of the intramolecular e lectron pathway, we have measured the midpoint potential values of the flav in cofactors and of the 3Fe-4S cluster of glutamate synthase in the isolate d alpha and beta subunits and in the alpha beta holoenzyme. No detectable a mounts 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 s ubunits to form the ap protomer does not alter significantly the midpoint p otential 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 bo und to the holoenzyme). Binding of the nonreducible NADP(+) analogue, 3-ami nopyridine adenine dinucleotide phosphate, made the measured midpoint poten tial 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 alp ha beta holoenzyme. This result correlates with the formation of a stable c harge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated beta subunit, but not in the alpha beta holoenz yme. Binding of L-methionine sulfone, a glutamine analogue, had no signific ant effect on the redox properties of the enzyme cofactors. On the contrary , 2-oxoglutarate made the measured midpoint potential value of the 3Fe-4S c luster approximately 20 mV more negative in the isolated alpha subunit, but up to 100 mV less negative in the alpha beta holoenzyme as compared to the values of the corresponding free enzyme forms, These findings are consiste nt 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 glutamat e synthase holoenzyme, but not in the isolated subunits, Furthermore, the d ata demonstrate a specific role of 2-oxoglutarate in promoting electron tra nsfer from FAD to the 3Fe-4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxoglutarate is indeed consistent with the recentl y determined three-dimensional structure of the glutamate synthase alpha su bunit, in which several polypeptide stretches are suitably positioned to me diate communication between substrate binding sites and the enzyme redox ce nters (FMN and the 3Fe-4S cluster) to tightly control and coordinate the in dividual reaction steps [Binda, C,, et al. (2000) Structure 8, 1299-1308].