Clavaminate synthase (CS) is one member of a large class of non-heme iron enzymes that require alpha-ketoglutarate (alpha-KG) as a cosubstrate. While the majority of this class catalyzes the hydroxylation of unactivated C-H bonds, CS is unusual in that in addition to performing hydroxylation chemistry, it also catalyzes the key oxidative ring closure and desaturation steps in the biosynthetic pathway to the potent beta-lactamase inhibitor clavulanic acid. A single non-heme Fe2+ site is responsible for all three of these reactions (hydroxylation, oxidative ring closure, and desaturation), during which 1 equiv of alpha-KG per reaction is decarboxylated into succinate and CO2, We have applied circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies to probe the geometric and electronic structure of the ferrous active site in the isozyme CS2 and its interaction with (alpha-KG. CD titration experiments show stoichiometric binding of Fe2+ to the apoenzyme, either with or without alpha-KG, as well as stoichiometric binding of alpha-KG to the iron containing enzyme. However, in the absence of the metal, the alpha-KG binding constant is reduced, indicating that Fe2+ facilitates cosubstrate binding at the active site. Ligand field CD and MCD data show that resting CS2 contains a six-coordinate ferrous center (10Dq = 10 050 cm(-1) Delta(5)E(g) = 1690 cm(-1)) and that addition of alpha-KG perturbs the site to produce a different six-coordinate center (10Dq = 9500 cm(-1), Delta(5)E(g) = 1630 cm(-1)). VTVH MCD analysis finds a ground-state splitting for resting CS2 (Delta(5)T(2g) approximate to -400 cm(-1)) that is fairly typical of six-coordinate ferrous sites, but a much larger splitting for CS2 + alpha-KG (Delta(5)T(2g) approximate to -1000 cm(-1)), indicative of Fe2+-alpha-KG pi interactions. UV/vis absorption, CD, and MCD spectroscopies have been applied to further probe the interaction of the cosubstrate with the metalloenzyme. These data show the appearance of low-lying metal-to-ligand charge-transfer transitions which demonstrate that alpha-KG binds directly to the iron. Furthermore, analysis and comparison to model complex data support a bidentate binding mode of alpha-KG, indicating that cosubstrate displaces two ligands from the six-coordinate resting active site to form a new six-coordinate alpha-KG-bound Fe2+ site. These results provide the first direct spectroscopic information about the nature of the CS2 ferrous active site and its interaction with alpha-KG and lend insight into the mechanism of this multifunctional enzyme.