Electron-Transfer Proteins Tweaked

Two research teams recently reported new ways to modify the properties of the copper-based electron-transfer protein azurin: replacing residues to change the oxidation-reduction (redox) potential in a controllable way, and modifying the copper center to enhance reactivity. The approaches might make it easier to adapt azurin and similar proteins for applications in artificial photosynthesis or in fuel cells for motor vehicles.

Redox processes power various biological and chemical phenomena ranging from photosynthesis and respiration to industrial catalysis and fuel cells. In nature, such processes are mediated by redox-active proteins such as azurin, a member of the cupredoxin family.

Just as a variety of batteries are needed to power different electronic devices, proteins with varied redox potentials are needed to drive different biological processes. Scientists would like to modify such proteins in a controlled way to exploit them in redox processes. But engineering redox proteins hasn’t been easy because a detailed understanding of how specific amino acid changes affect redox potentials and other protein properties has been elusive.

Redox potentials of copper complexes have previously been modified by changing solvents or pH. But changing solvents would be impractical for applications like fuel cells, and solvent and pH changes can cause undesired effects on the nonredox properties of proteins, such as their stability and how efficiently they transfer electrons.

Now, metalloprotein design and engineering specialist Yi Lu of the University of Illinois, Urbana-Champaign, and coworkers report having succeeded at tweaking the potential of azurin in a predictable manner and over a wide range without solvent or pH changes (Nature 2009, 462, 113). They did this by modifying amino acids so as to change hydrophobic and hydrogen-bonding interactions just outside the protein’s active site. The research team was able to tune the potential of azurin over a 700-mV range in water, surpassing both the lowest and highest potentials ever reported for any natural or engineered cupredoxin protein.

The researchers found that increasing the hydrophobicity of amino acid residues near the active site increases the redox potential, and changing hydrogen-bond interactions among residues either decreases or increases it. A key finding of the study is that the changes in redox potential resulting from such residue modifications are additive, which has been hypothesized but never before demonstrated.

The work advances a fundamental understanding of noncovalent interactions in redox proteins and could also lead to proteins with tailored redox potentials for various applications. It has previously been difficult, if not impossible, to vary the potentials of redox proteins in water without varying other electron-transfer properties in unwanted ways, Lu notes.

The new azurin variants “will find a wide range of applications as redox agents and electron-transfer reagents in water, where the reduction potential can be changed while keeping surface interactions with redox partners and electron-transfer pathways the same,” Lu says. He and his coworkers hope to show that the approach is applicable to other redox proteins as well.

“Although people have been using engineered azurins for many years to understand how the protein environment influences the reduction potential of copper centers, controlled tuning over a wide range has not been achieved,” comments metalloprotein structure and function authority Amy C. Rosenzweig of Northwestern University. Mutating proteins is not novel, but Lu and coworkers “combined mutations in a very smart way,” she says, “and it is pretty amazing that the effects of different mutations are additive. This work really teaches us something about how proteins tune redox potential and will impact future protein design work.”

“That a 700-mV range can be achieved with substitutions at only three sites is remarkable and opens the possibility of adapting azurin as an electron donor to new enzyme systems,” says Michael Murphy, a researcher at the University of British Columbia whose work focuses on metal-based enzymes.

The study “has provided a quantitative scale that will be very helpful to those of us in the field,” adds electron-transfer expert Harry B. Gray of California Institute of Technology.

Indeed, Gray and coworkers have been engineering azurin in a completely different way. In an independent study, they report synthesizing an azurin with a new class of copper center (Nat. Chem., DOI: 10.1038/nchem.412).

In natural proteins, mononuclear copper centers are classified as type 1 or 2 by differences in spectroscopic properties, structure, and function. Gray’s group has now created modified bacterial azurins that are “type zero”—so called because they are fundamentally different from either previously known type. The modified proteins have enhanced electron-transfer reactivities relative to the type-2 azurin on which they’re based.

By making simple changes in the coordination sphere of azurin copper centers, Gray and coworkers created “something unique that does not fit the long-standing classification scheme,” Rosenzweig says. “Type zero may find uses in catalytic applications, and it may even exist in biology”—a suspicion that has yet to be confirmed.

Overall, the results from both Lu’s and Gray’s studies “are encouraging for the future of metalloprotein design,” Rosenzweig says.