De novo proteins as models of radical enzymes

Catalytically essential side-chain radicals have been recognized in a growi ng number of redox enzymes. Here we present a novel approach to study this class of redox cofactors. Our aim is to construct a de novo protein, a radi cal maquette, that will provide a protein framework in which to investigate how side-chain radicals are generated, controlled, and directed toward cat alysis. A tryptophan and a tyrosine radical maquette, denoted alpha(3)W(1) and alpha(3)Y(1), respectively, have been synthesized, alpha(3)W(1) and alp ha(3)Y(1) contain 65 residues each and have molecular masses of 7.4 kDa. Th e proteins differ only in residue 32, which is the position of their single aromatic side chain. Structural characterization reveals that the proteins fold in water solution into thermodynamically stable, alpha-helical confor mations with well-defined tertiary structures. The proteins are resistant t o pH changes and remain stable through the physiological pH range. The arom atic residues are shown to be located within the protein interior and shiel ded from the bulk phase, as designed. Differential pulse voltammetry was us ed to examine the reduction potentials of the aromatic side chains in alpha (3)W(1) and alpha(3)Y(1) and compare them to the potentials of tryptophan a nd tyrosine when dissolved in water. The tryptophan and tyrosine potentials were raised considerably when moved from a solution environment to a well- ordered protein milieu. We propose that the increase in reduction potential of the aromatic residues buried within the protein, relative to the soluti on potentials, is due to a lack of an effective protonic contact between th e aromatic residues and the bulk solution.