Azospirillum brasilense glutamate synthase (GltS) is a complex iron–sulfur flavoprotein whose catalytically active αβ protomer (α subunit, 162 kDa; β subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe–4S]0, +1, and two [4Fe–4S]+1, +2 clusters. The structure of the α subunit has been determined providing information on the mechanism of ammonia transfer from l -glutamine to 2-oxoglutarate through a 30 Å-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe–S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe–4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS β subunit with alanyl residues. The engineered genes encoding the β subunit variants (and derivatives carrying C-terminal His6 -tags) were co-expressed with the wild-type α subunit gene. In all cases the C/A substitutions prevented α and β subunits association to yield the GltS αβ protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe–4S]+1, +2 clusters within the N-terminal region of the β subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in αβ heterodimer formation by structuring an N-terminal [Fe–S] β subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal β subunit-like domain.