5′-deoxyadenosine contacts the substrate radical intermediate in the active site of ethanolamine ammonia-lyase: ²H and ¹³C electron nuclear double resonance studies

The mechanism of propagation of the radical center between the cofactor, substrate, and product in the adenosylcobalamin- (AdoCbl) dependent reaction of ethanolamine ammonia-lyase has been probed by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The radical of S-2-aminopropanol, which appears in the steady state of the reaction, was used in ENDOR experiments to determine the nuclear spin transition frequencies of ²H introduced from either deuterated substrate or deuterated coenzyme and of ¹³C introduced into the ribosyl moiety of AdoCbl. A ²H doublet (1.4 MHz splitting) was observed centered about the Larmor frequency of ²H. Identical ENDOR frequencies were observed for ²H irrespective of its mode of introduction into the complex. A ¹³C doublet ENDOR signal was observed from samples prepared with [U-¹³C-ribosyl]-AdoCbl. The ¹³C coupling tensor obtained from the ENDOR powder pattern shows that the ¹³C has scalar as well as dipole-dipole coupling to the unpaired electron located at C1 of S-2-aminopropanol. The dipole-dipole coupling is consistent with a distance of 3.4 ± 0.2 Å between C1 of the radical and C5′ of the labeled cofactor component. These results establish that the C5′ carbon of the 5′-deoxyadenosyl radical moves ∼7 Å from its position as part of AdoCbl to a position where it is in contact with C1 of the substrate which lies ∼12 Å from the Co²⁺ of cob(II)alamin. These findings are also consistent with the contention that 5′-deoxyadenosine is the sole mediator of hydrogen transfers in ethanolamine ammonia-lyase.