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. 2012 Jun 4;51(23):5589-92.
doi: 10.1002/anie.201201981. Epub 2012 Apr 26.

A designed functional metalloenzyme that reduces O2 to H2O with over one thousand turnovers

Kyle D Miner  1 , Arnab Mukherjee, Yi-Gui Gao, Eric L Null, Igor D Petrik, Xuan Zhao, Natasha Yeung, Howard Robinson, Yi Lu
Affiliations

A designed functional metalloenzyme that reduces O2 to H2O with over one thousand turnovers

Kyle D Miner et al. Angew Chem Int Ed Engl. .

Abstract

Rational design of functional enzymes with high turnovers is a significant challenge, especially those with complex active site and difficult reactions, such as in respiratory oxidases. Introducing 2 His and 1 Tyr into myoglobin resulted in designed enzymes that reduce O2 to H2O with > 1000 turnovers and minimal release of reactive oxygen species. This also showed that presence and positioning of Tyr, not Cu, are critical for activity.

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Figures

Figure 1
Figure 1
Structures and computer models of native and designed oxidases. a) Overlay of the crystal structures of bovine CcO (grey) and E-F33Y-CuBMb (cyan); b) Overlay of the crystal structure of cbb3 HCO from Pseudomonas stutzeri[10] (tan) and E-G65Y-CuBMb computer model (yellow); The CuB copper in CcO is represented as an orange sphere.
Figure 2
Figure 2
Characterization of product by 17O NMR spectroscopy; normalized area of the H217O peak for WTswMb (gray), E-F33Y-CuBMb (red) and E-G65Y-CuBMb (blue) at 30, 60, 90, and 120 minutes. Error bars indicate standard deviation.
Figure 3
Figure 3
Characterization of the O2 reduction; a) Rates of oxygen reduction to form either water (blue) or superoxide/peroxide (red) with 18 µM WTswMb, CuBMb, F33Y-CuBMb or G65Y-CuBMb; b) Number of turnovers of O2 reduction by E-F33Y-CuBMb (black) and E-G65Y-CuBMb (red); blue arrow indicates addition of ~28 equivalents O2. Inset, region of high number of turnovers.
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