The use of highly reactive, electron-deficient radical intermediates
to perform biologically-challenging reactions is an emergent theme in enzyme
catalysis. In the family of vitamin B12 coenzyme- (adenosylcobalamin-)
dependent enzymes, substrate binding-triggered thermal cleavage of the
cobalt-carbon bond of B12 generates low spin (S=1/2) CoII and the 5'-deoxyadenosyl
organic radical. In the proposed minimal mechanism, the deoxyadenosyl
radical abstracts a hydrogen atom from the bound substrate to form a substrate
radical, which rearranges to a product radical. Thus, a sequence
of different CoII-radical pair states exists during the radical-mediated
steps of catalysis. The structure and dynamics of these states in
ethanolamine deaminase from Salmonella typhimurium are revealed by using
techniques of X-band continuous-wave and high-resolution pulsed-electron
paramagnetic resonance spectroscopy, in combination with specific 2H-,
15N- and 13C-labeled reactants. We have directly identified, for
the first time in any B12 system, a product radical intermediate, and propose
the contribution of a "product radical trap" to radical pair yield in Class
II B12 enzymes. Electron spin echo envelope modulation (ESEEM) spectroscopy
is used to pinpoint the relative positions of the 5'-carbon deoxyadenosine
and the substrate-based unpaired spin. The CoII ligation in catalytically-engaged
enzyme is characterized and compared to model cobalamin by using 14N ESEEM.
The results merge to provide insight into the principles of high-yield
radical pair generation in vitamin B12 coenzyme-dependent enzymes.