E1催化的最初的脱水反应是由阴离子诱导的，这个阴离子由维生素B6 PMP稳定(Scheme 5).
中间体22是由来自于E3 的 NADH 还原，生成中间体23。而[2Fe-2S]存在于E1E3中，负责单电子转移。
存在于E3的FAD, 被NADH还原，可以储存两个电子，而隔离于 E3 的[2Fe-2S] 中心。这两个电子最终通过E1的[2Fe-2S] 中心还原中间体22。接下来希夫碱23的水解释放出来C3脱氧的化合物11，重生PMP，结束E1-E3催化。氢负离子从NADH转移到FAD 形成被两个电子还原的醌(Scheme 5).
和其他FNR家族的黄素蛋白类似，E3中的FAD经历两步，分别还原E1和E3结合的[2Fe-2S]。E1E3的氧化还原中心，是 一个模块化的方式。E3的铁硫簇失去后，FAD依然可以被NADH还原。 E1的铁硫簇失去后，不会阻止脱水生成中间体22。
E3含有一个序列保守的植物类型的黄素蛋白。E1中的CXnCX1CX7C 序列，在其他的 [2Fe-2S] 蛋白中并不常见. 因此，这个序列代表了一个催化C3位脱氧的合作结构域。
中间体22结合在E1的活性中心，E1 E3之间有密切的联系来进行电子转移。实际上，E1E3复合物在FPLC中观察到。酵母双杂交实验也证明二者的相互作用。二者的解离常数是288nM, 比其他黄素蛋白还原酶的弱。尽管E1E3的辅酶是独立工作的，在电子传递和存储中，他们交流紧密。从E3移除铁硫簇，严重影响等电点。
EPR检测来监测E1-E3的催化反应。并发现了两个自由基中间体。一个是黄素醌类，一个是底物10相关的自由基。在 g 2.003 缺少自由基的指纹信号，证明在中间体24中，具有PMP上的单电子，而非 3，4糖上 (Scheme 5)。反应中间体可能是葡萄糖-PMP 复合物。
2.2.6 STUDIES ON THE LOCALIZATION OF THE UNPAIRED ELECTRON SPIN IN THE E1–E3
MECHANISM The role of the PMP cofactor in the radical-based E1–E3 reactions
is unprecedented since most PMP/PLP-dependent enzymes employ anionic
chemistry in their catalysis. Thus several isotopically labeled PMP analogs, including [4,5-2H4]PMP and [2-C2H3]PMP, were prepared to probe the location of the single-electron spin (102). When [4,5-2H4]PMP-reconstituted E1
was used in the E1-E3 catalysis, the radical signal was found to be narrowed by approximately 3 G as compared to that of the reference spectrum. Such a deuterium-induced sharpening effect on the EPR signal is indicative of threplacement of a strongly hyperfine-coupled 1H by 2H. The Mims ENDOR(electron nuclear double resonance) spectra of the radical signals from the incubation of substrate and E1 reconstituted with either PMP or its deuteriumlabeled analogs suggested that the free electron spin can reside in at least two
positions on the PMP ring (24b and 24d), which couples with each of the two sets of deuterium labels (4/5-2H4 and 2-2H3) and gives a moderately large 2H hyperfine EPR signal. This is by far the most conclusive evidence for the
formation of an unprecedented PMP-based radical in the mechanism of the E1–E3
catalysis. The participation of PMP in a deoxygenation reaction is uncommon,
but it is the direct involvement of PMP in a radical-based reduction that truly
places E1 in a class by itself.