The alternative oxidase (AOX) is a monotopic di‑iron carboxylate protein which acts as a terminal respiratory chain oxidase in a variety of plants, fungi and protists. Of particular importance is the finding that both emerging infectious diseases caused by human and plant fungal pathogens, the majority of which are multi-drug resistant, appear to be dependent upon AOX activity for survival. Since AOX is absent in mammalian cells, AOX is considered a viable therapeutic target for the design of specific fungicidal and anti-parasitic drugs.

In this work, we have mutated conserved residues within the hydrophobic channel (R96, D100, R118, L122, L212, E215 and T219), which crystallography has indicated leads to the active site. Our data shows that all mutations result in a drastic reduction in V_max and catalytic efficiency whilst some also affected the K_m for quinol and oxygen. The extent to which mutation effects inhibitor sensitivity was also investigated, with mutation of R118 and T219 leading to a complete loss of inhibitor potency. However, only a slight reduction in IC₅₀ values was observed when R96 was mutated, implying that this residue is less important in inhibitor binding. In silico modelling has been used to provide insight into the reason for such changes, which we suggest is due to disruptions in the proton transfer network, resulting in a reduction in overall reaction kinetics. We discuss our results in terms of the structural features of the ubiquinol binding site and consider the implications of such findings on the nature of the catalytic cycle.

Significance

The alternative oxidase is a ubiquinol oxidoreductase enzyme that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. It is widely distributed amongst the plant, fungal and parasitic kingdoms and plays a central role in metabolism through facilitating the turnover of the TCA cycle whilst reducing ROS production.

中文翻译：

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泛醇结合位点的动力学和结构表征以及锥虫替代氧化酶的氧还原作用。

替代氧化酶（AOX）是一种单峰二铁羧酸盐蛋白，在多种植物，真菌和原生生物中充当末端呼吸链氧化酶。特别重要的发现是，由人类和植物真菌病原体引起的新兴传染病似乎都依赖于AOX活性才能生存，而人类和植物真菌病原体大多具有多重耐药性。由于哺乳动物细胞中不存在AOX，因此AOX被认为是设计特定的杀菌和抗寄生虫药物的可行治疗靶标。

在这项工作中，我们已经突变了疏水通道（R96，D100，R118，L122，L212，E215和T219）中的保守残基，这些晶体学表明它们导致了活性位点。我们的数据表明，所有突变均会导致V _max和催化效率急剧降低，而某些突变还影响了喹诺醇和氧的K _m。还研究了突变影响抑制剂敏感性的程度，R118和T219突变导致抑制剂效能完全丧失。但是，IC ₅₀仅略有减少当R96突变时，观察到该值，表明该残基在抑制剂结合中不那么重要。在计算机模拟中，已使用建模方法来洞察此类变化的原因，我们认为这是由于质子转移网络的破坏，导致整体反应动力学降低。我们讨论了泛醇结合位点的结构特征方面的结果，并考虑了这些发现对催化循环性质的影响。

意义

备选的氧化酶是泛醇氧化还原酶，其催化泛醇的氧化和将氧气还原为水。它广泛分布在植物，真菌和寄生虫王国中，并且通过促进三氯乙酸循环的周转同时减少活性氧的产生，在代谢中起着核心作用。