Alternative oxidase (AOX) catalyzes the four-electron reduction of dioxygen to water as an additional terminal oxidase, and the catalytic reaction is critical for the parasite to survive in its bloodstream form. Recently, the X-ray crystal structure of trypanosome alternative oxidase (TAO) complexed with ferulenol was reported and the molecular structure of the non-heme diiron center was determined. The binding of O₂ was a unique side-on type compared to other iron proteins. In order to characterize the O₂ binding state of TAO, the O₂ binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O₂ binding state is the end-on type, and the binding states of the side-on type are higher in energy. Based on the binding energies and electronic structure analyses, O₂ binds very weakly to the TAO iron center (ΔE =6.7 kcal mol⁻¹) in the electronic state of Fe(II)…OO, not in the suggested charge transferred state such as the superoxide state (Fe(III)OO· ^–) as seen in hemerythrin. Coordination of other ligands such as water, Cl⁻, CN⁻, CO, N₃⁻ and H₂O₂ was also examined, and H₂O₂ was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol⁻¹. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by H₂O₂ in a dose-dependent and reversible manner.

替代氧化酶（AOX）催化双氧四电子还原为水，作为另一种末端氧化酶，并且催化反应对于寄生虫以其血流形式存活至关重要。最近，报道了与阿魏烯醇复合的锥虫替代氧化酶（TAO）的X射线晶体结构，并确定了非血红素二铁中心的分子结构。与其他铁蛋白相比，O ₂的结合是独特的侧面结合类型。为了表征将O ₂ TAO的结合状态时，O ₂结合的状态在本研究中一个量子力学/分子力学（QM / MM）的理论水平进行检索。我们发现最稳定的O ₂结合状态是端接型和侧接型的结合态的能量较高。根据结合能和电子结构分析，O ₂以Fe（II）…O O的电子态非常弱地结合到TAO铁中心（ΔE = 6.7 kcal mol ^-1），而不是建议的电荷转移态像是在红藻素中看到的超氧化物态（Fe（III）O O· ^–）。其它配体，例如水，氯的协调^-，CN ^-，CO，N ₃ ^-和H ₂ ö ₂也进行了检查，且H ₂ ö ₂发现ΔE＝ 14.0kcal mol ^-1与Fe（II）位点最牢固地结合。通过测量TAO和小隐隐孢子虫AOX的泛醇氧化酶活性，通过实验证实了这一点，发现该活性被H ₂ O ₂抑制，呈剂量依赖性和可逆性。