FIG 3.

LC-MS profiles of the reaction catalyzed by Pno using purified EtfAB-I as the sole electron acceptor in the presence of both H₂¹⁶O and H₂¹⁸O. The reaction mixture contained 20 mM phosphate buffer (pH 7.4), 1 mM pseudooxynicotine, 89 μM purified EtfAB-I, 0.03 μM purified Pno, and 40% (vol/vol) H₂¹⁸O. (A) LC profile of the reaction, where an enlargement of peak 3 is shown in the inset. (1) Mass spectrum of N-methylmyosmine (m/z 161.1101), which was the product of spontaneous dehydration reaction from the substrate pseudooxynicotine. There is a hydrolysis equilibrium between N-methylmyosmine and pseudooxynicotine. (2) Mass spectra of the excess substrate pseudooxynicotine (m/z 179.1211) and its dehydrated product N-methylmyosmine (m/z 161.1103) with retention time at 3.8 min. (3) Mass spectra of the product 3-succinoyl-semialdehyde-pyridine (m/z 164.0735 for ¹⁶O-labeled, m/z 166.0778 for ¹⁸O-labeled) and its adduct of water (m/z 182.0842 for ¹⁶O-labeled, m/z 184.0882 for ¹⁸O-labeled). The ratio of the two peak areas for ¹⁶O- and ¹⁸O-labeled products (∼1.5) is identical to the ratio of H₂¹⁶O and H₂¹⁸O in the reaction mixture.