Abstract：
Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.
摘要：
来自空肠弯曲杆菌的周质硝酸还原酶NapA（C.jejuni）包含钼辅因子（Moco）和4Fe-4S簇，并催化硝酸盐还原为亚硝酸盐。催化所需的还原当量从NapC→NapB→NapA转移。从NapB到NapA的电子转移通过NapA中的4Fe-4S团簇发生。空肠杆菌NapA在Mo-辅因子和4Fe-4S簇之间具有保守的赖氨酸(K79)。K79与4Fe-4S簇形成H键相互作用，并通过H键网络将后者与Moco连接。因此，可以想象，K79可能在分子内电子转移和NapA的催化活性中发挥重要作用。在本研究中,我们发现K79突变为Ala导致活性几乎完全丧失，表明其在催化活性中的作用。氰化物对空肠弯曲菌NapA的抑制作用,硫氰酸盐,和叠氮化物也被调查。抑制研究表明，氰化物以非竞争性方式抑制NapA，而硫氰酸盐和叠氮化物以无竞争性的方式抑制NapA。两种抑制机制都不涉及抑制剂与Mo中心的直接结合。已经在NapAK79A变体的催化活性丧失的背景下讨论了这些结果,并且已经提出了NapA中可能的阴离子结合位点。
