Spectroscopic Studies of Substrate Interactions with Clavaminate Synthase 2, a Multifunctional α-KG-Dependent Non-Heme Iron Enzyme: Correlation with Mechanisms and Reactivities

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Abstract
Using a single ferrous active site, clavaminate synthase 2 (CS2) activates O2 and catalyzes the hydroxylation of deoxyguanidinoproclavaminic acid (DGPC), the oxidative ring closure of proclavaminic acid (PC), and the desaturation of dihydroclavaminic acid (and a substrate analogue, deoxyproclavaminic acid (DPC)), each coupled to the oxidative decarboxylation of cosubstrate, α-ketoglutarate (α-KG). CS2 can also catalyze an uncoupled decarboxylation of α-KG both in the absence and in the presence of substrate, which results in enzyme deactivation. Resting CS2/FeII has a six-coordinate FeII site, and α-KG binds to the iron in a bidentate mode. The active site becomes five-coordinate only when both substrate and α-KG are bound, the latter still in a bidentate mode. Absorption, CD, MCD, and VTVH MCD studies of the interaction of CS2 with DGPC, PC, and DPC provide significant molecular level insight into the structure/function correlations of this multifunctional enzyme. There are varying amounts of six-coordinate ferrous species in the substrate complexes, which correlate to the uncoupled reaction. Five-coordinate ferrous species with similar geometric and electronic structures are present for all three substrate/α-KG complexes. Coordinative unsaturation of the FeII in the presence of both cosubstrate and substrate appears to be critical for the coupling of the oxidative decarboxylation of α-KG to the different substrate oxidation reactions. In addition to the substrate orientation relative to the open coordination position on the iron site, it is hypothesized that the enzyme can affect the nature of the reactivity by further regulating the binding energy of the water to the ferrous species in the enzyme/succinate/product complex.