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Physical Chemistry

Chemists Mutate Protein To Achieve Wider-Than-Ever Redox Range

Molecular Design: Protein variants have expanded electron-transfer ability

by Stu Borman
December 2, 2015 | A version of this story appeared in Volume 93, Issue 48

Structure shows amino acid mutations that permit azurin to adopt a range of redox potential values, which are shown on a scale.
Credit: Proc. Nat. Acad. Sci. USA
Researchers varied the metal (teal sphere) and mutated five side chains (ball-and-stick figures) in native azurin (Cu-WTAz) to create several versions with a range of redox potentials (see scale). F114N changes phenylalanine (F) to asparagine (N) at sequence position 114, and so on. Red modifications increase redox potential, and blue changes lower it.

Some proteins in the body have the ability to transfer electrons to or accept them from other molecules. How readily one of these proteins takes on new electrons is indicated by its so-called redox potential, measured in volts. Now researchers have modified a protein to tune its electron-transfer ability across a 2-V range—the first time variants of a single protein have been designed to adopt such a wide range of redox potentials.

The achievement shows that scientists are beginning to understand protein structure-function effects at a detailed enough level to control redox behavior completely. This capability could ease the way to improved redox enzymes that controllably activate C–H bonds in organic reactions and to advanced redox reagents for fuel cells and solar energy converters.

To reach such a wide redox range, Yi Lu of the University of Illinois, Urbana-Champaign, and coworkers rationally mutated several amino acids around the metal center in a bacterial protein called azurin. They also swapped out the metal in the site. The changes created azurins that adopt redox potentials from +0.972 to –0.950 V (Proc. Natl. Acad. Sci. USA 2015, DOI: 10.1073/pnas.1515897112). These voltages span nearly the entire physiological redox potential range, which (relative to a common redox standard) goes from about +1 V, where water is oxidized, to –1 V, where H3O+ is reduced to H2.

Natural azurin (called WTAz) has a copper cofactor and a potential of +0.265 V. Lu and coworkers shifted its potential to +0.972 V by creating HPAz, an azurin with three mutations that increase Cu-binding site hydrophobicity and two mutations that modify key hydrogen-bonding interactions. To reach –0.950 V, they replaced Cu with Ni and made a mutation that added negative charge to the metal site. They then used different combinations of the same mutational and cofactor changes to reach five other redox potentials along the scale.

“Demonstrating the ability to tune redox potential over a range of 2 V is remarkable,” comments Victor L. Davidson of the University of Central Florida, an expert on enzymatic electron transfer. “Many studies have demonstrated that redox potentials of proteins can be altered by site-directed mutagenesis and metal substitution. But I don’t think anyone believed it would be possible to span this large a range of redox potentials in a single protein using these two approaches.”

Lu says the tuned azurins can be used as water-soluble redox agents in many biochemical studies, and his group is now trying to tune redox potentials of other metalloenzymes as well. “We have filed a patent and are in the process of finding potential licensees,” he says.

This article has been translated into Spanish by and can be found here.



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