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.
Keywords: Binding energy; End-on; Inhibitor; Potential energy surface; QM/MM; Side-on.
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