The Wang Lab studies oxygen activation and C–H/C–C bond functionalization by metalloenzymes. Molecular oxygen is a powerful oxidant, but its reaction with ground-state singlet molecules is kinetically unfavorable due to its spin-forbidden nature. To harness the oxidizing power of oxygen and manipulate biomolecules, iron-containing enzymes are frequently employed by aerobic organisms. Heme and non-heme iron-dependent oxygenases represent two of the most ubiquitous and potent natural catalysts, while the contribution of the ligand sets to oxygen activation and reaction outcome remains largely unknown due to the massive variations between heme and non-heme systems. We aim to fill the knowledge gap through a comparative investigation of the biomedically essential oxygenases with specific ligand sets. Structural and spectroscopic techniques are used to probe the catalytic mechanisms and structure-function correlations of these systems. Our research will broaden the understanding of iron-oxygen chemistry and establish a platform for therapeutic interventions.