Non-Coordinative Binding of O2 at the Active Center of a Copper-Dependent Enzyme.

Molecular oxygen (O2 ) is a sustainable oxidation reagent. O2 is strongly oxidizing but kinetically stable and its final reaction product is water. For these reasons learning how to activate O2 and how to steer its reactivity along desired reaction pathways is a longstanding challenge in chemical research.[1] Activation of ground-state diradical O2 can occur either via conversion to singlet oxygen or by one-electron reduction to superoxide. Many enzymes facilitate activation of O2 by direct fomation of a metal-oxygen coordination complex concomitant with inner sphere electron transfer. The formylglycine generating enzyme (FGE) is an unusual mononuclear copper enzyme that appears to follow a different strategy. Atomic-resolution crystal structures of the precatalytic complex of FGE demonstrate that this enzyme binds O2 juxtaposed, but not coordinated to the catalytic CuI . Isostructural complexes that contain AgI instead of CuI or nitric oxide instead of O2 confirm that formation of the initial oxygenated complex of FGE does not depend on redox activity. A stepwise mechanism that decouples binding and activation of O2 is unprecedented for metal-dependent oxidases, but is reminiscent of flavin-dependent enzymes.

Structure of formylglycine-generating enzyme in complex with copper and a substrate reveals an acidic pocket for binding and activation of molecular oxygen.

The formylglycine generating enzyme (FGE) catalyzes oxidative conversion of specific peptidyl-cysteine residues to formylglycine. FGE mediates O2-activation and hydrogen-atom abstraction in an active site that contains Cu(i) coordinated to two cysteine residues. Similar coordination geometries are common among copper-sensing transcription factors and copper-chaperone but are unprecedented among copper-dependent oxidases. To examine the mechanism of this unusual catalyst we determined the 1.04 Å structure of FGE from Thermomonospora curvata in complex with copper and a cysteine-containing peptide substrate. This structure unveils a network of four crystallographic waters and two active site residues that form a highly acidic O2-binding pocket juxtaposed to the trigonal planar tris-cysteine coordinated Cu(i) center. Comparison with structures of FGE in complex with Ag(i) and Cd(ii) combined with evidence from NMR spectroscopy and kinetic observations highlight several structural changes that are induced by substrate binding and prime the enzyme for O2-binding and subsequent activation.