A large number of enzymes are now known that use carbon-based radicals to catalyze a variety of unusual and chemically difficult reactions. Whereas in free solution such reactive radical species have extremely short life times and react very non-specifically, when generated at the active site of an enzyme they can be very stable and catalyze remarkably specific reactions. We are interested in how enzymes generate free radicals and harness their intrinsic reactivity to towards productive catalysis.

We are studying glutamate mutase, which catalyzes a 'simple', but highly unusual, carbon skeleton rearrangement of L-glutamate to L-threo-3-methylaspartate as part of the glutamate fermentation pathway in various anaerobic bacteria. In this reaction a hydrogen on carbon-4 of glutamate is interchanged with the glycyl group on carbon-3 to give methylaspartate. Glutamate mutase is one of a group of enzymes that catalyse unusual rearrangement reactions that involve radical intermediates. These enzymes use the cofactor adenosylcobalamin (coenzyme-B₁₂, a biologically active form of vitamin B₁₂) to generate an adenosyl radical through homolysis of the coenzyme's unique cobalt-carbon bond.

The adenosyl radical generated by B₁₂ is used to remove the migrating hydrogen from the substrate, in this case glutamate, to form a substrate radical, a step common to all B₁₂ isomerases. This radical rearranges to form a product radical, in this case methylaspartyl radical, and then the hydrogen is replaced from the coenzyme to give methylaspartate and regenerate the adenosyl radical which may then be 'stored' by reforming the cobalt-carbon bond. In essence, the introduction of the unpaired electron onto the substrate serves to activate it towards chemical reactions that would not otherwise be feasible.

We have shown that the enzyme accelerates homolysis of the coenzyme by a factor of one trillion fold! Furthermore, generation of adenosyl radical and removal of hydrogen from the substrate are closely coordinated events. Thus, sensibly, the enzyme never forms radicals unless the substrate is bound. We have also shown that the rearrangement of glutamyl radical to methylaspartyl radical occurs by fragmentation of the glutamyl radical, to give acrylate and a glycyl radical as intermediates, followed by recombination of the glycyl radical with the other end of the acrylate double bond to yield the methylaspartyl radical. We have also investigated the free energy profile of the overall reaction and uncovered evidence for quantum tunneling of the migrating hydrogen atoms during the reaction.