Exploring amino-acid radicals and quinone redox chemistry in model proteins

Abstract: 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 (?3W) and a 117-residue four-helix bundle (?4W). ?3W and ?4W are single-chain and uniquely structured proteins. They are redox inert except for a single radical site (position 32 in ?3W and 106 in ?4W). Papers I and II describe the design process and the protein characteristics of ?4W as well as a voltammetry study of its unique tryptophan. Paper III and V describe two projects based on ?3C, which is a Trp-32 to Cys-32 variant of ?3W. In Paper III we use ?3C 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 ?3C system is extended in Paper V to study quinone redox chemistry. Papers III and V contain protocols to generate the cofactor-containing ?3C systems and descriptions of their protein properties. Paper IV describes efforts to redesign ?3Y (a Trp-32 to Tyr-32 variant of ?3W) 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.