Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme.Fe(II).2OG.substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual 'ping-pong' mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penicillin N ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.