Carbon–hydrogen (C–H) bonds are the foundation of essentially every organic molecule, making them an ideal place to do chemical synthesis. The key challenge is achieving selectivity for one particular C(sp3)−H bond1–3. In recent years, metalloenzymes have been found to perform C(sp3)−H bond functionalization4,5. Despite substantial progresses in the past two decades6,7, enzymatic halogenation and pseudohalogenation of unactivated C(sp3)−H—providing a functional handle for further modification—have been achieved with only non-haem iron/α-ketoglutarate-dependent halogenases, and are therefore limited by the chemistry possible with these enzymes8. Here we report the discovery and characterization of a previously unknown halogenase ApnU, part of a protein family containing domain of unknown function 3328 (DUF3328). ApnU uses copper in its active site to catalyse iterative chlorinations on multiple unactivated C(sp3)−H bonds. By taking advantage of the softer copper centre, we demonstrate that ApnU can catalyse unprecedented enzymatic C(sp3)−H bond functionalization such as iodination and thiocyanation. Using biochemical characterization and proteomics analysis, we identified the functional oligomeric state of ApnU as a covalently linked homodimer, which contains three essential pairs—one interchain and two intrachain—of disulfide bonds. The metal-coordination active site in ApnU consists of binuclear type II copper centres, as revealed by electron paramagnetic resonance spectroscopy. This discovery expands the enzymatic capability of C(sp3)−H halogenases and provides a foundational understanding of this family of binuclear copper-dependent oxidative enzymes.
