Weak O binding and strong HO binding at the non-heme diiron center of trypanosome alternative oxidase.

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 O was a unique side-on type compared to other iron proteins. In order to characterize the O binding state of TAO, the O binding states were searched at a quantum mechanics/molecular mechanics (QM/MM) theoretical level in the present study. We found that the most stable O 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, O binds very weakly to the TAO iron center (ΔE =6.7 kcal mol) 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, N and HO was also examined, and HO was found to bind most strongly to the Fe(II) site by ΔE = 14.0 kcal mol. This was confirmed experimentally through the measurement of ubiquinol oxidase activity of TAO and Cryptosporidium parvum AOX which was found to be inhibited by HO in a dose-dependent and reversible manner.