Burns K D, Pieper P A, Liu H W, Stankovich M T
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Biochemistry. 1996 Jun 18;35(24):7879-89. doi: 10.1021/bi960284t.
Studies of the biosynthesis of ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis V, have shown that the C-3 deoxygenation is a process consisting of two enzymatic steps. The first enzyme involved in this transformation is CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is a pyridoxamine 5'-phosphate dependent iron-sulfur protein. The second catalyst, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase, formally called CDP-6-deoxy-delta(3,4)-glucoseen reductase (E3), is an NADH dependent plant type [2Fe-2S] containing flavoenzyme. To better understand the electron transfer carried out by these two enzymes, the potentials of the E1 and E3 redox cofactors were determined spectroelectrochemically. At pH 7.5, the midpoint potential of the E3 FAD was found to be -212 mV, with the FADox/FADsq couple (E1o') and the FADsq/FADhq couple (E2o') calculated to be -231 and -192 mV, respectively. However, the E1o' and E2o' of the FAD in E3(apoFeS) at pH 7.5 were estimated to be -215 and -240 mV, respectively, which are quite different from those of the holo-E3, suggesting a significant effect of the iron-sulfur center on the redox properties of the flavin coenzyme. Our data also showed that the midpoint potential of the E3 iron-sulfur is -257 mV and that of the E1 [2Fe-2S] center is -209 mV. These values indicated a thermodynamic barrier to the proposed electron transfer of NADH->FAD=>E3[2Fe-2S]->E1[2Fe-2S] at pH 7.5. Regulation of electron transfer by several mechanisms is possible and experiments were performed to examine ways of overcoming the unfavorable electron transfer energetics in the E1/E3 system. It was found that both binding of E3 with NAD+ and complex formation between E3 and E1 showed no effect on the midpoint potentials of the E3 FAD and iron-sulfur center. Interestingly, the midpoint potential of the E3 FAD shifts dramatically to -273 mV (E1o' approximately -345 mV and E2o' approximately -200 mV) at pH 8.4, with very little semiquinone stabilization (< 5%). The potential of the E3 [2Fe-2S] center at pH 8.4 was also found to undergo a negative shift to -279 mV, and that of the E1 iron sulfur center remained essentially the same at -206 mV. These data indicated that the redox properties of this system may be regulated by pH and the electron transfer between the E3 redox centers may be prototropically controlled. These results also demonstrated that E3 is unique among this class of enzymes.
对鼠李糖（一种在假结核耶尔森氏菌V型脂多糖中发现的3,6 - 二脱氧己糖）生物合成的研究表明，C - 3脱氧是一个由两个酶促步骤组成的过程。参与此转化的第一种酶是CDP - 6 - 脱氧 - L - 苏式 - D - 甘油 - 4 - 己酮糖 - 3 - 脱水酶（E1），它是一种依赖磷酸吡哆胺5'-磷酸的铁硫蛋白。第二种催化剂，CDP - 6 - 脱氧 - L - 苏式 - D - 甘油 - 4 - 己酮糖 - 3 - 脱水酶还原酶，正式名称为CDP - 6 - 脱氧 - δ(3,4) - 葡萄糖烯还原酶（E3），是一种依赖NADH的含植物型[2Fe - 2S]的黄素酶。为了更好地理解这两种酶进行的电子转移，通过光谱电化学法测定了E1和E3氧化还原辅因子的电位。在pH 7.5时，发现E3黄素腺嘌呤二核苷酸（FAD）的中点电位为 - 212 mV，FADox/FADsq偶联（E1o'）和FADsq/FADhq偶联（E2o'）分别计算为 - 231和 - 192 mV。然而，在pH 7.5时，E3（脱辅基铁硫蛋白）中FAD的E1o'和E2o'分别估计为 - 215和 - 240 mV，这与全酶E3的电位有很大差异，表明铁硫中心对黄素辅酶的氧化还原性质有显著影响。我们的数据还表明，E3铁硫的中点电位为 - 257 mV，E1 [2Fe - 2S]中心的中点电位为 - 209 mV。这些值表明在pH 7.5时，所提出的从NADH到FAD再到E3[2Fe - 2S]到E1[2Fe - 2S]的电子转移存在热力学障碍。通过几种机制调节电子转移是可能的，并且进行了实验以研究克服E1/E3系统中不利电子转移能量学的方法。发现E3与NAD + 的结合以及E3和E1之间的复合物形成对E3 FAD和铁硫中心的中点电位均无影响。有趣的是，在pH 8.4时，E3 FAD的中点电位急剧变为 - 273 mV（E1o'约为 - 345 mV，E2o'约为 - 200 mV），半醌稳定化程度非常小（<5%）。还发现pH 8.4时E3 [2Fe - 2S]中心的电位负移至 - 279 mV，而E1铁硫中心的电位在 - 206 mV基本保持不变。这些数据表明该系统的氧化还原性质可能受pH调节，并且E3氧化还原中心之间的电子转移可能受质子转移控制。这些结果还证明E3在这类酶中是独特的。
