Jepson Brian J N, Anderson Lee J, Rubio Luis M, Taylor Clare J, Butler Clive S, Flores Enrique, Herrero Antonia, Butt Julea N, Richardson David J
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
J Biol Chem. 2004 Jul 30;279(31):32212-8. doi: 10.1074/jbc.M402669200. Epub 2004 May 27.
Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a 4Fe-4S cluster. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (E(m)) of -190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g(av) = 2.0047 in air-oxidized enzyme that accounted for only 10-20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g(av) = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of -100 to -350 mV (E(m) Mo(6+/5+) = -150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below -200 mV, where the cofactors are predominantly 4Fe-4S and Mo(5+). The data suggests that during the catalytic cycle nitrate will bind to the Mo(5+) state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.
细菌细胞质同化型硝酸还原酶是所有硝酸还原酶亚组中特征最不清楚的。在本研究中，通过分光电位滴定法和蛋白质膜伏安法对蓝藻聚球藻属PCC 7942的铁氧化还原蛋白依赖性硝酸还原酶NarB进行了分析。金属和酸不稳定硫化物分析显示，NarB分子的铁/硫/钼最接近的整数值为4:4:1。通过低温电子顺磁共振对连二亚硫酸盐还原酶的分析表明，在10 K时存在一个4Fe-4S簇特征的信号。NarB的电子顺磁共振监测电位滴定表明，该簇作为n = 1的能斯特组分滴定，中点氧化还原电位（E(m)）为-190 mV。在60 K收集的电子顺磁共振光谱显示，在空气氧化的酶中存在一个称为“非常高g值”的Mo(V)信号，g(av) = 2.0047，仅占总钼的10-20%。用连二亚硫酸盐还原后，该信号消失，并观察到一个新的“高g值”物种（g(av) = 1.9897）。在电位滴定中，高g值的Mo(V)信号在-100至-350 mV的电位范围内出现（E(m) Mo(6+/5+) = -150 mV），当完全出现时，它占每摩尔酶1摩尔的Mo(V)。NarB的蛋白质膜伏安法表明，在低于-200 mV的电位下活性开启，此时辅因子主要是4Fe-4S和Mo(5+)。数据表明，在催化循环中，硝酸盐将与NarB的Mo(5+)状态结合，此时酶最少被双电子还原。将NarB的光谱特性与膜结合和周质呼吸型硝酸还原酶的光谱特性进行比较，发现它与周质酶密切相关，但NarB钼中心的电位被调节为在较低电位下运行，这与NarB与细胞质中低电位铁氧化还原蛋白的偶联一致。
