Converting triplet dioxygen into a powerful oxidant is fundamentally important to life. We will show that thiolate (RS–) ligands facilitate this process with iron by lowering the activation barrier to O2 binding, and favoring the formation of an RS-Fe(III)-O2•– intermediate that is capable of cleaving strong C-H bonds.1 We will also show experimentally for the first time that an inner-sphere mechanism is involved in RS-Fe(III)-O2•– formation. We will show using T-dependent stopped-flow kinetics that H-bond donors and ligand constraints can alter the reversibility of, and kinetic barrier to, O2 binding,2 demonstrating the huge impact that a secondary coordination sphere and protein constraints can have on a metalloenzyme. We will also experimentally and computationally show that oxo atom transfer to a cis thiolate sulfur involves a stepwise, as opposed to a concerted, mechanism involving a high-valent iron oxo containing an antiferromagnetically coupled radical that is delocalized over two thiolate sulfurs. Mossbauer and EPR evidence to support the involvement of a metastable high-valent iron oxo will also be presented. Thiolate-ligated Fe(III)-O2•– intermediates are involved in key biological processes such as the biosynthesis of β-lactam antibiotics, as well as the regulation of cysteine, toxic levels of which can lead to neurological disorders, or metastases of cancerous tumors.