Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate-dependent nonheme ferrous...

Determining the requirements for efficient oxygen (O₂) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O₂) and a sequential mechanism (cofactor binding and reaction with O₂ precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the Fe^II), αKG binding to Fe^II-DAOCS results in ∼45% five-coordinate sites that selectively react with O₂ relative to the remaining six-coordinate sites. However, this reaction produces an Fe^III species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to Fe^II-DAOCS produces five-coordinate sites that rapidly react with O₂ to form an Fe^IV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.