Engineered Proteins: Redox Properties and Their Applications

Oxidoreductases and metalloproteins, representing more than one third of all known proteins, serve as significant catalysts for numerous biological processes that involve electron transfers such as photosynthesis, respiration, metabolism, and molecular signaling. The functional properties of the oxidoreductases/metalloproteins are determined by the nature of their redox centers. Protein engineering is a powerful approach that is used to incorporate biological and abiological redox cofactors as well as novel enzymes and redox proteins with predictable structures and desirable functions for important biological and chemical applications. The methods of protein engineering, mainly rational design, directed evolution, protein surface modifications, and domain shuffling, have allowed the creation and study of a number of redox proteins. This review presents a selection of engineered redox proteins achieved through these methods, resulting in a manipulation in redox potentials, an increase in electron-transfer efficiency, and an expansion of native proteins by de novo design. Such engineered/modified redox proteins with desired properties have led to a broad spectrum of practical applications, ranging from biosensors, biofuel cells, to pharmaceuticals and hybrid catalysis. Glucose biosensors are one of the most successful products in enzyme electrochemistry, with reconstituted glucose oxidase achieving effective electrical communication with the sensor electrode; direct electron-transfer-type biofuel cells are developed to avoid thermodynamic loss and mediator leakage; and fusion proteins of P450s and redox partners make the biocatalytic generation of drug metabolites possible. In summary, this review includes the properties and applications of the engineered redox proteins as well as their significance and great potential in the exploration of bioelectrochemical sensing devices. Antioxid. Redox Signal. 17, 1796–1822. I. Introduction A. Redox properties of naturally occurring/wild-type proteins 1. Metalloproteins a. Iron/heme proteins b. Copper metalloproteins c. Zinc-finger proteins d. Other metal-based (nickel, vanadium, and molybdenum) redox proteins 2. Flavoproteins 3. Quinoproteins 4. Redox-active amino-acid residue proteins B. Electron transfer in proteins II. Engineering Redox-Active Proteins A. Methods available for designing artificial redox proteins 1. Rational design 2. Directed evolution 3. Domain shuffling/molecular Lego 4. Catalytic antibodies 5. Protein reconstitution 6. Incorporation of non-natural redox residues/metallocofactors B. Electrochemical methods that characterize engineered redox proteins III. Properties of Engineered Redox Proteins A. Engineered copper proteins B. Engineered heme proteins C. Engineered FeS cluster proteins D. Engineered proteins containing redox active residues IV. Applications of Engineered Redox Proteins A. Biosensors 1. Glucose biosensors 2. Iron-based redox biosensors B. Biofuel cells C. Photovoltaic cells D. Electrochemical immunoassay E. Pharmaceutical applications of engineered redox proteins F. Engineered redox proteins in catalysis V.