<p>Amino-acid radical enzymes have been studied extensively for 30 years but the experimental barriers to determine the thermodynamic properties of their key radical cofactors are so challenging that only a handful of reports exist in the literature. This is a major drawback when trying to understand the long-range radical transfer and/or catalytic mechanisms of this important family of enzymes. Here this issue is addressed by developing a library of well-structured model proteins specifically designed to study tyrosine and tryptophan radicals. The library is based on a 67-residue three-helix bundle (α₃W) and a 117-residue four-helix bundle (α₄W). α₃W and α₄W are single-chain and uniquely structured proteins. They are redox inert except for a single radical site (position 32 in α₃W and 106 in α₄W). Papers I and II describe the design process and the protein characteristics of α₄W as well as a voltammetry study of its unique tryptophan. Paper III and V describe two projects based on α₃C, which is a Trp-32 to Cys-32 variant of α₃W. In Paper III we use α₃C to investigate what effect the degree of solvent exposure of the phenolic OH group has on the redox characteristics of tyrosine analogs. We show that the potential of the PhO•/PhOH redox pair is dominated by interactions with the OH group and that the environment around the hydrophobic part of the phenol has no significant impact. In addition, we observe that interactions between the phenolic OH group and the protein matrix can raise the phenol potential by 0.11-0.12 V relative to solution values. The α₃C system is extended in Paper V to study quinone redox chemistry. Papers III and V contain protocols to generate the cofactor-containing α₃C systems and descriptions of their protein properties. Paper IV describes efforts to redesign α₃Y (a Trp-32 to Tyr-32 variant of α₃W) to contain an interacting Tyr-32/histidine pair. The aim is to engineer and study the effects of a redox-induced proton acceptor in the Tyr-32 site.</p>

Amino-acid radical enzymes have been studied extensively for 30 years but the experimental barriers to determine the thermodynamic properties of their key radical cofactors are so challenging that only a handful of reports exist in the literature. This is a major drawback when trying to understand the long-range radical transfer and/or catalytic mechanisms of this important family of enzymes. Here this issue is addressed by developing a library of well-structured model proteins specifically designed to study tyrosine and tryptophan radicals. The library is based on a 67-residue three-helix bundle (α3W) and a 117-residue four-helix bundle (α4W). α3W and α4W are single-chain and uniquely structured proteins. They are redox inert except for a single radical site (position 32 in α3W and 106 in α4W). Papers I and II describe the design process and the protein characteristics of α4W as well as a voltammetry study of its unique tryptophan. Paper III and V describe two projects based on α3C, which is a Trp-32 to Cys-32 variant of α3W. In Paper III we use α3C to investigate what effect the degree of solvent exposure of the phenolic OH group has on the redox characteristics of tyrosine analogs. We show that the potential of the PhO•/PhOH redox pair is dominated by interactions with the OH group and that the environment around the hydrophobic part of the phenol has no significant impact. In addition, we observe that interactions between the phenolic OH group and the protein matrix can raise the phenol potential by 0.11-0.12 V relative to solution values. The α3C system is extended in Paper V to study quinone redox chemistry. Papers III and V contain protocols to generate the cofactor-containing α3C systems and descriptions of their protein properties. Paper IV describes efforts to redesign α3Y (a Trp-32 to Tyr-32 variant of α3W) to contain an interacting Tyr-32/histidine pair. The aim is to engineer and study the effects of a redox-induced proton acceptor in the Tyr-32 site.

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