Characterization of the Product Radical Structure in the CoII-Product Radical Pair State of Coenzyme B12-Dependent Ethanolamine Deaminase by Using Three-Pulse2H ESEEM Spectroscopy

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Abstract
Molecular structural features of the product radical in the CoII-product radical pair catalytic intermediate state in coenzyme B12- (adenosylcobalamin-) dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band three-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The CoII-product radical pair state was prepared by cryotrapping holoenzyme during steady-state turnover on excess 1,1,2,2-2H4-aminoethanol or natural abundance, 1H4-aminoethanol. Simulation of the 2H/1H quotient ESEEM (obtained at two microwave frequencies, 8.9 and 10.9 GHz) from the interaction of the unpaired electron localized at C2 of the product radical with nearby 2H nuclei requires four types of coupled 2H, which are assigned as follows: (a) a single strongly coupled (effective dipole distance, reff = 2.3 Å) 2H in the C5‘ methyl group of 5‘-deoxyadenosine, (b) two weakly coupled (reff = 4.2 Å) 2H in the C5‘ methyl group, (c) one 2H coupling from a β-2H bonded to C1 of the product radical (isotropic hyperfine coupling, Aiso = 4.7 MHz), and (d) a second type of C1 β-2H coupling (Aiso = 7.7 MHz). The two β-2H couplings are proposed to arise from two C1−C2 rotamer states of the product radical that are present in approximately equal proportion. A model is presented, in which C5‘ is positioned at a distance of 3.3 Å from C2, which is comparable with the C1−C5‘ distance in the CoII-substrate radical pair intermediate. Therefore, the C5‘ methyl group remains in close (van der Waals) contact with the substrate and product radical species during the radical rearrangement step of the catalytic cycle, and the C5‘ center is the sole mediator of radical pair recombination in ethanolamine deaminase.