Identification of the 4-glutamyl radical as an intermediate in the carbon skeleton rearrangement catalyzed by coenzyme B12-dependent glutamate mutase from Clostridium cochlearium

A series of H-2- and C-13-labeled glutamates were used as substrates for coenzyme B-12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-H-2], [3,3-H-2(2)], [4,4-H-2(2)], [2,3,3,4,4-H-2(5)], [2-C-13], [3-C-13], and [4-C-13]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g(xy)' = 2.1, g(z)' = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(IT)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 Angstrom. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical, These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B-12.