A mechanistic study of H2O2-dependent C-H bond hydroxylation by myoglobin reconstituted with a manganese porphycene was carried out. The X-ray crystal structure of the reconstituted protein obtained at 1.5 A resolution reveals tight incorporation of the complex into the myoglobin matrix at pH 8.5, the optimized pH value for the highest turnover number of hydroxylation of ethylbenzene. The protein generates a spectroscopically detectable two-electron oxidative intermediate in a reaction with peracid, which has a half-life up to 38 s at 10 degrees C. Electron paramagnetic resonance spectra of the intermediate with perpendicular and parallel modes are silent, indicating formation of a low-spin Mn-V-oxo species. In addition, the Mn-V-oxo species is capable of promoting the hydroxylation of sodium 4-ethylbenzenesulfonate under single turnover conditions with an apparent second-order rate constant of 2.0 M-1 s(-1) at 25 degrees C. Furthermore, the higher bond dissociation enthalpy of the substrate decreases the rate constant, in support of the proposal that the H-abstraction is one of the rate-limiting steps. The present engineered myoglobin serves as an artificial metalloenzyme for inert C-H bond activation via a high-valent metal species similar to the species employed by native monooxygenases such as cytochrome P450.