Weak O₂ binding and strong H₂O₂ binding at the non-heme diiron center of trypanosome alternative oxidase

Highlights

• Trypanosome alternative oxidase (TAO) contains a non-heme diiron center.

• The binding energy of O₂ is calculated to be weak (6.7 kcal mol⁻¹).

• O₂ binding state of TAO is end-on.

• The binding energy of H₂O₂ is strongest (14.0 kcal mol⁻¹).

• H₂O₂ can inhibit the O₂ binding process of TAO.

Abstract

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.