Circular Dichroism and Magnetic Circular Dichroism Spectroscopic Studies of the Non-Heme Ferrous Active Site in Clavaminate Synthase and Its Interaction with α-Ketoglutarate Cosubstrate

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Abstract

Clavaminate synthase (CS) is one member of a large class of non-heme iron enzymes that require α-ketoglutarate (α-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 β-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 α-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 α-KG. CD titration experiments show stoichiometric binding of Fe2+ to the apoenzyme, either with or without α-KG, as well as stoichiometric binding of α-KG to the iron-containing enzyme. However, in the absence of the metal, the α-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, Δ5Eg = 1690 cm-1) and that addition of α-KG perturbs the site to produce a different six-coordinate center (10Dq = 9500 cm-1, Δ5Eg = 1630 cm-1). VTVH MCD analysis finds a ground-state splitting for resting CS2 (Δ5T2g ≈ −400 cm-1) that is fairly typical of six-coordinate ferrous sites, but a much larger splitting for CS2 + α-KG (Δ5T2g ≈ −1000 cm-1), indicative of Fe2+−α-KG π 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 α-KG binds directly to the iron. Furthermore, analysis and comparison to model complex data support a bidentate binding mode of α-KG, indicating that cosubstrate displaces two ligands from the six-coordinate resting active site to form a new six-coordinate α-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 α-KG and lend insight into the mechanism of this multifunctional enzyme.

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