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47 WWW.CEN-ONLINE.ORG NOVEMBER 16, 2009 SCIENCE & TECHNOLOGY TWO RESEARCH TEAMS recently re- ported new ways to modify the properties of the copper-based electron-transfer pro- tein 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 biologi- cal and chemical phenomena ranging from photosynthesis and respiration to indus- trial 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 pro- cesses. 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 de- tailed 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 chang- ing 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 engi- neering specialist Yi Lu of the University of Illinois, Urbana-Champaign, and cowork- ers 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-bond- ing 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 in- teractions among residues either decreases or increases it. A key finding of the study is that the changes in redox potential result- ing from such residue modifications are additive, which has been hypothesized but never before demonstrated. The work advances a fundamental understand- ing of noncovalent interac- tions in redox proteins and could also lead to proteins with tailored redox poten- tials for various applications. It has previously been dif- ficult, if not impossible, to vary the potentials of redox proteins in water without varying other electron- transfer properties in un- wanted ways, Lu notes. The new azurin variants “will find a wide range of applications as redox agents and electron-transfer re- agents in water, where the reduction potential can be changed while keeping sur- face interactions with redox partners and electron-trans- fer pathways the same,” Lu says. He and his coworkers hope to show that the ap- proach is applicable to other redox proteins as well. “Although people have been using engineered azurins for many years to un- derstand how the protein environment influences the reduction potential of cop- per centers, controlled tuning over a wide range has not been achieved,” comments metalloprotein structure and function au- thority Amy C. Rosenzweig of Northwest- ern 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 pro- teins 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 differ- ent way. In an independent study, they re- port synthesizing an azurin with a new class of copper center ( Nat. Chem., DOI: 10.1038/nchem.412). In natural proteins, mononuclear copper cen- ters are classified as type 1 or 2 by differences in spectroscopic properties, structure, and function. Gray’s group has now created modified bacte- rial azurins that are “type zero”—so called because they are fundamentally dif- ferent 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 appli- cations, 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,” Rosen- zweig says. —STU BORMAN COURTESY OF NICHOLAS MARSHALL & YI LU PROTEINS BY DESIGN Lu and coworkers changed the redox potential of azurin by modifying key amino acids (shown as stick representations). Cu is the green sphere. ELECTRON-TRANSFER PROTEINS TWEAKED Modifications for BETTER CONTROL of redox potential and reactivity
