Engendering Catalytic Activity By Increasing Dynamics In A Designed Enzyme

Designed proteins often differ from natural proteins in their extraordinary stability and their low level of dynamic behavior. Such dynamic motion is an underappreciated but critical aspect of functional computational protein design. Here we report the rational redesign of DF2, an extremely rigid, computationally designed homodimeric diiron protein, into the functional enzyme CDM13, a single-chain dimetal-containing four-helix bundle, by increasing the dynamic motion of the protein via the reversal of the amino acid sequence of two of the helices, greatly reducing the packing complementarity in the protein core. The diiron form of CDM13 activates oxygen, forming μ-oxo-bridged diferric clusters at a rate 33 times greater than DF2, at a kcat of .117 per minute versus a kcat of .0035 per minute. This form also reacts with 4-aminophenol, producing benzoquinone monoimine with a kcat of .02 per minute. The reduced form of dimanganese-containing CDM13 oxidizes hydrogen peroxide by two electrons at a rate greater than 1000 s(-1), more than 10000 times faster than the oxidized form reduces it to water. Thus, CDM13 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. The ability of these enzymes to catalyze the oxidation of methane to methanol will also be discussed. The creation of such a broad range of catalytic activity in a multifunctional active site like the dimetal site of DF2 merely by increasing dynamic motion underscores the need to move beyond the paradigm of structure in protein design to encompass a more realistic view that encompasses protein and enzyme dynamics.