The essential cofactor of the enzyme ribonucleotide reductase is a stable tyrosyl free radical adjacent to a carboxylate- and oxide- bridged diiron(III) cluster. This cofactor is the target of existing anticancer and antiviral drugs and is an attractive target for design of new pharmaceuticals. The cofactor can assemble spontaneously into the enzymes from Escherichia coli and mammals when they are exposed to Fe(II) ions and molecular oxygen. The assembly reaction comprises binding of Fe(II) ions by the protein and subsequent reaction of its diiron(II) cluster with O2 to form the tyrosyl radical and diiron(III) cluster. The oxygen binding/tyrosyl radical formation phase of the reaction is quite complex and poorly understood. The reaction has similarities to the catalytic reactions of structurally-related proteins such as methane monooxygenase and stearoyl acyl carrier protein delta desaturase. Many believe that similar intermediate diiron structures form in each of these reactions. If so, each protein must interact with the common diiron intermediate(s) to guide the reaction down the appropriate mechanistic pathway to the desired outcome. In no case have the details of these interactions been fully discovered. We have constructed a series of engineered ribonucleotide reductase proteins that interact with different diiron intermediates formed in their reactions with O2 in very distinct and interesting ways. The mechanistic characterization of the O2 reactions of these proteins and the structural characterization of their diiron intermediates that we propose to undertake should indicate which particular features of the natural ribonucleotide reductase protein are important for it to ensure the desired reaction outcome in preference to those that occur in the other diiron proteins. Understanding this control should suggest vulnerabilities that might be exploited for drug design. In addition, the characterization of engineered proteins that do carry out other reactions should prove instructive in the design of synthetic diiron clusters that can also mediate the interesting and important oxidation reactions carried out by members of this grown class of proteins.
Showing the most recent 10 out of 18 publications
