Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I–expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I–overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2—an enzyme without a counterpart in mammals—as a candidate target for leishmanicidal drugs.

II 型 NADH 脱氢酶 (NDH2) 是存在于线粒体内膜外表面或内表面的单位酶，通过将电子从 NADH 传递到泛醌而无需耦合质子易位，从而有助于 NADH/NAD+ 平衡。在这里，我们将存在于人类原生动物寄生虫利什曼原虫的所有物种中的基因的产物描述为真正的、面向基质的 II 型 NADH 脱氢酶。在线粒体内，这种呼吸活动与某些利什曼原虫物种中的 I 型 NADH 脱氢酶（复合物 I）的呼吸活动一致，但在其他物种中则不然。为了质疑 NDH2 在寄生虫生理学中的重要性，我们尝试了它在两种寄生虫物种中的遗传破坏，表现出沉默的（婴儿利什曼原虫，李）和完全可操作的（主要利什曼原虫，Lm) 复合体 I。引人注目的是，该分析显示，利什曼原虫不能耐受 NDH2 消除，即使是表达复合体 I 的 Lm 物种也不耐受。相反，如果 NDH2 充分表达，复合物 I 在这两个物种中都是可有可无的。即使在存在活性复合物 I 的情况下，II 型脱氢酶也是必不可少的，这将利什曼原虫NADH 代谢置于一个完全独特的视角，并表明 NDH2 的未探索功能超越了其复杂的 I 重叠活动。值得注意的是，通过证明 NDH2 的基本特征延伸到利什曼原虫的致病阶段，我们在基因上验证了 NDH2（一种在哺乳动物中没有对应物的酶）作为杀利什曼原虫药物的候选靶标。