Understanding How the Protein Environment Accelerates Cofactor‐Free O2 Activation in Antibiotic Biosynthesis Monooxygenases (ABMs)

Abstract

Molecular oxygen is a nontoxic, naturally abundant, thermodynamically powerful oxidant. Most enzyme‐catalyzed oxidation and oxygenation processes using O2 depend on small, tightly bound metal‐containing or organic cofactors, such as heme or flavin, to activate O2 and thereby accelerate its reaction with organic substrates. Antibiotic Biosynthesis Monooxygenases (ABMs) are a large family of small proteins with the unusual ability to catalyze direct reactions between their substrates and O2 without any cofactors. Prior studies of a representative ABM, Nogalamycin MonoOxygenase (NMO), suggested that its reaction proceeds by rate limiting electron transfer to form a superoxide/substrate radical pair. By comparing the rate and thermodynamic parameters for the catalyzed and uncatalyzed reactions, we sought to determine how the protein environment of NMO helps to catalyze the direct reaction between its substrate and O2. Marcus Theory relates the activation energy (ΔG‡, the difference in energy between the substrate and transition state) to the thermodynamic driving force (ΔG°, the difference in energy between substrate and product) and the reorganization energy (λ0, the energy required to reorganize all atomic nuclei in the reaction sphere without electron transfer): ΔG‡ =(ΔG° + λ0)2/4λ0. Prior work showed that for the flavin‐containing enzyme glucose oxidase, the reorganization energy was responsible for most of the difference in ΔG‡ between the enzymatic and uncatalyzed electron transfers that occur between flavin and O2. A single conserved positive charge was essential for lowering ΔG‡ and therefore accelerating the reaction. We hypothesized that λ0 might likewise be the major contributor to ΔΔG‡ (catalyzed versus uncatalyzed) in NMO, though these enzymes lack conserved positive charges. Here, we tested that hypothesis by measuring ΔG° (via spectroelectrochemical titration of the midpoint potentials) and ΔG‡ (Arrhenius plots) for uncatalyzed and NMO‐catalyzed oxygenation reactions, as well as for reactions catalyzed by two key NMO active site mutants. Our results suggest an unconventional means for accelerating reactions between O2 and certain classes of substrate that depends solely on a properly tuned protein environment.Support or Funding InformationNational Science Foundation, MCB1715176This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.