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. 2015 Sep 16;137(36):11570-3.
doi: 10.1021/jacs.5b07119. Epub 2015 Sep 8.

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Yang Yu, Chang Cui, Xiaohong Liu  1 , Igor D Petrik, Jiangyun Wang  1 , Yi Lu
Affiliations

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Yang Yu et al. J Am Chem Soc. .

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.

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Figures

Figure 1
Figure 1
(a) Structures of G65Y-CuBMb(+6), showing the engineered lysines in blue, and cyt b5 (PDB IDs 1CYO for cyt b5(3) and 4FWY for F33Y-CuBMb; rendered through VMD). (b) Oxidase activity of G65Y-CuBMb(+6) in comparison with those of native cyt cbb3 oxidase and G65Y-CuBMb at the same concentration under the typical conditions of: NADH, 2 mM; cyt b5 reductase, 80 nM; cyt b5, 5 μM; G65Y-CuBMb(+6), 50 nM. The black arrows indicate the addition of reductant and the double arrow shows the injection of native cyt cbb3 oxidase.
Figure 2
Figure 2
Studies of ET between cyt b5 and G65Y-CuBMb or G65YCuBMb(+6). (a) Representative stopped-flow UV–vis spectra of cyt b5 oxidation catalyzed by G65Y-CuBMb(+6) and (inset) time traces at characteristic wavelengths. (b) Time traces of cyt b5 oxidation represented by the absorption at 556 nm, overlaid with global spectroscopic fitting (red lines).
Figure 3
Figure 3
(a) Measurement of O2 reduction and H2O2 production in the O2 reduction reaction. (b) Representative kinetic UV–vis spectra of reaction solution with 100 μM NADH, sampled at 0.5 s intervals over 100 s. The inset shows NADH oxidation and oxygen reduction catalyzed by G65Y-CuBMb(+6).
See this image and copyright information in PMC

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