A kinetic and spectroscopic characterization of the ferryl interme-diate (APO-II) from APO, the heme-thiolate peroxygenase fromAgrocybe aegerita,is described. APO-II was generated by reactionof the ferric enzyme with metachloroperoxybenzoic acid in thepresence of nitroxyl radicals and detected with the use of rapid-mixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxylradicals served as selective reductants of APO-I, reacting only slowlywith APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nmand 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pKaof 10.0 for theFeIV−O−H of APO-II, indicating that APO-II is protonated under typ-ical turnover conditions. Kinetic characterization showed that APO-IIis unusually reactive toward a panel of benzylic C−H and phenolicsubstrates, with second-order rate constants for C−HandO−Hbond scission in the range of 10–107M−1·s−1. Our results demon-strate the important role of the axial cysteine ligand in increasingthe proton affinity of the ferryl oxygen of APO intermediates, thusproviding additional driving force for C−HandO−H bond scission.