Engendering Methane Monooxygenase And Hydrogen Peroxide Oxidase Activity Into A Designed Dimetal Protein By Increasing Protein Dynamics

It has become relatively easy to design proteins which are extremely rigid but lack the degree of dynamic motion required for catalytic activity. Here we report the rational redesign of DF2, an extremely rigid, computationally designed homodimeric diiron protein, into two functional enzymes - a methane monooxygenase when bound to two iron ions and a hydrogen peroxide oxidase when bound to two manganese ions. This was achieved by simply reversing of the amino acid sequence of two of the helices, greatly reducing the packing complementarity in the protein core - both decreasing the protein stability and greatly increasing the dynamic motion of the enzyme. The resulting diiron enzyme catalyzes the hydroxylation of methane to methanol when supplied either with molecular oxygen and reducing equivalents or hydrogen peroxide. The dimanganese enzyme oxidizes hydrogen peroxide by two electrons forming molecular oxygen at a rate of at least 400 s−1 while only catalyzing the reduction of hydrogen peroxide to water at a rate of 0.08 s−1. Most natural enzymes with peroxide as a substrate are catalases - hydrogen peroxide dismutases which catalyze both reactions at high rates in order to lower the cellular concentration of hydrogen peroxide. Because it favors the oxidizing half-reaction over the other by a factor of 5000, dimanganese CDM3 promises to serve as a unique alternate version of an oxygen evolving cluster in the design of new photoautotrophic organisms which perform carbon fixation utilizing light and low energy electrons originating from what is typically considered to be a toxic intercellular waste product.