A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Abstract

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

Design and engineering of artificial oxygen-activating metalloenzymes

“…b 5 ), the G65Y-Cu B Mb variant was shown to reduce oxygen with rates (52 s −1 ) comparable to a native C c O (50 s −1 ). 228 Moreover, when the designed protein was immobilized onto electrodes, the electrocatalytic reduction of oxygen using G65Y-Cu B Mb not only resulted in very selective oxygen reduction (~ 96% water) but also exhibited rates (5000 s −1 ) exceeding the fastest CcO (bovine CcO with rates of 500 s −1 ). 229 These results demonstrated that it is possible to redesign the metal-binding sites in native scaffolds that display new functions (e.g., from reversible O 2 binding in WTMb to catalytic O 2 reduction to H 2 O) that can meet or even exceed the catalytic efficiency of native enzymes.…”

Design of Heteronuclear Metalloenzymes

“…b 5 (Figure 8a) reduced oxygen at a rate (52 s −1 ) comparable to that of a native HCO (cyt. cbb 3 oxidase, 50 s −1 )(Yu et al, 2015). …”

“…© 2014 American Chemical Society (c) Enhancement of binding affinity and catalytic efficiency of MnC c P mimics by mutation secondary coordination sphere residues. Figures are adapted with permission from ref (Yu et al, 2015), (Bhagi-Damodaran et al, 2014) and (Hosseinzadeh et al, 2016) respectively.…”

Advances in Engineered Hemoproteins that Promote Biocatalysis

“…By incorporating a copper binding site and changing the electrostatics of the solvent accessible sites, the L29H/F43H/G65Y and D44K/D60K/E85K mutant of myoglobin transformed the oxygen storage protein into a robust oxidase with dramatic enhanced functionality. A few such studies incorporated a Cu B site in myoglobin in order to provide insight into the mechanism of reduction of dioxygen by heme-copper oxidases [8,[51][52][53][54]. In this way, myoglobin serves as a small model protein that mimics the large membrane-bound heme-copper oxidase.…”