1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyzes the last step in the biosynthesis of the gaseous plant hormone ethylene, which is involved in development, including germination, fruit ripening, and senescence. ACCO is a mononuclear non-heme ferrous enzyme that couples the oxidation of the cosubstrate ascorbate to the oxidation of substrate ACC by dioxygen. In addition to substrate and cosubstrate, ACCO requires the activator CO2 for continuous turnover. NIR circular dichroism and magnetic circular dichroism spectroscopies have been used to probe the geometric and electronic structure of the ferrous active site in ACCO to obtain molecular-level insight into its catalytic mechanism. Resting ACCO/FeII is coordinatively saturated (six-coordinate). In the presence of CO2, one ferrous ligand is displaced to yield a five-coordinate site only when both the substrate ACC and cosubstrate ascorbate are bound to the enzyme. The open coordination position allows rapid O2 activation for the oxidation of both substrates. In the absence of CO2, ACC binding alone converts the site to five-coordinate, which would react with O2 in the absence of ascorbate and quickly deactivate the enzyme. These studies show that ACCO employs a general strategy similar to other non-heme iron enzymes in terms of opening iron coordination sites at the appropriate time in the reaction cycle and define the role of CO2 as stabilizing the six-coordinate ACCO/FeII/ACC complex, thus preventing the uncoupled reaction that inactivates the enzyme.