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.

Figure 1

(a) Structures of G65Y-Cu_BMb(+6), showing the engineered lysines in blue, and cyt b₅ (PDB IDs 1CYO for cyt b₅(3) and 4FWY for F33Y-Cu_BMb; rendered through VMD). (b) Oxidase activity of G65Y-Cu_BMb(+6) in comparison with those of native cyt cbb₃ oxidase and G65Y-Cu_BMb at the same concentration under the typical conditions of: NADH, 2 mM; cyt b₅ reductase, 80 nM; cyt b₅, 5 μM; G65Y-Cu_BMb(+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

Studies of ET between cyt b₅ and G65Y-Cu_BMb or G65YCuBMb(+6). (a) Representative stopped-flow UV–vis spectra of cyt b₅ oxidation catalyzed by G65Y-Cu_BMb(+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

(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-Cu_BMb(+6).