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. 2016 Jan 12;113(2):262-7.
doi: 10.1073/pnas.1515897112. Epub 2015 Dec 2.

Design of a single protein that spans the entire 2-V range of physiological redox potentials

Parisa Hosseinzadeh  1 , Nicholas M Marshall  2 , Kelly N Chacón  3 , Yang Yu  1 , Mark J Nilges  4 , Siu Yee New  1 , Stoyan A Tashkov  5 , Ninian J Blackburn  6 , Yi Lu  7
Affiliations

Design of a single protein that spans the entire 2-V range of physiological redox potentials

Parisa Hosseinzadeh et al. Proc Natl Acad Sci U S A. .

Abstract

The reduction potential (E°') is a critical parameter in determining the efficiency of most biological and chemical reactions. Biology employs three classes of metalloproteins to cover the majority of the 2-V range of physiological E°'s. An ultimate test of our understanding of E°' is to find out the minimal number of proteins and their variants that can cover this entire range and the structural features responsible for the extreme E°'. We report herein the design of the protein azurin to cover a range from +970 mV to -954 mV vs. standard hydrogen electrode (SHE) by mutating only five residues and using two metal ions. Spectroscopic methods have revealed geometric parameters important for the high E°'. The knowledge gained and the resulting water-soluble redox agents with predictable E°'s, in the same scaffold with the same surface properties, will find wide applications in chemical, biochemical, biophysical, and biotechnological fields.

Keywords: azurin; cupredoxins; electron transfer; reduction potential; secondary coordination sphere.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
By mutating only five residues and using two metal ions (Cu and Ni) in a single protein, Az, the entire range of physiological potential can be covered. (A) Overall structure of Az. (B) The metal-binding site of azurin and the residues around the site investigated in this study. (C) WTAz active site structure, Protein Data Bank (PDB) ID code 4AZU. (D) Minimized structural model of HPAz. (E) Structure of M121E-Az, PDB ID code 4QLW. (F) Minimized structural model of N47S/F114N/M121L-Az.
Fig. 2.
Fig. 2.
Cyclic voltammogram showing the reversible redox couple of HPAz. The potential was obtained at scan rate of 0.1 V/s at pH 5.0 in 50 mM ammonium acetate buffer (black line). The signal with the capacitive current subtracted is shown as an inset in the middle (red line). (Em = 0.97 ± 0.02 V vs. SHE).
Fig. 3.
Fig. 3.
Stopped-flow UV-vis spectra of oxidation of 0.7 mM Cu(I)-HPAz by 1 eq Na2IrCl6 at pH 6.5 in 50 mM phosphate buffer and 100 mM NaCl. (A) Spectral changes in first 10 s of reaction. (B) Simulated spectra of individual species as determined by the SpecFit program. (C) Time-dependent transitions of Na2IrCl6, Blue Cu(II)-HPAz and Green Cu(II)-HPAz, as simulated by the SpecFit program.
Fig. 4.
Fig. 4.
EPR spectra of Na2IrCl6 oxidation of Cu(I)-HPAz. (Top) Spectrum shows freeze-quenched sample after 20 ms of the reaction. (Bottom) Spectrum is the final product of the reaction. The broad feature at values smaller than g = 2.0 in the 20-ms sample is due to packing of the tube during the freeze-quench process. Black line: experimental spectra; red line: simulated spectra.
Scheme 1.
Scheme 1.
Proposed mechanism of the oxidation of Cu(I)-HPAz with Na2IrCl6.
See this image and copyright information in PMC

Comment in

  • A single protein redox ruler.
    Bains RK, Warren JJ. Bains RK, et al. Proc Natl Acad Sci U S A. 2016 Jan 12;113(2):248-50. doi: 10.1073/pnas.1522425112. Epub 2015 Dec 16. Proc Natl Acad Sci U S A. 2016. PMID: 26676579 Free PMC article. No abstract available.

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