Three redox states of Trypanosoma brucei alternative oxidase identified by infrared spectroscopy and electrochemistry

Abstract

Electrochemistry coupled with Fourier transform infrared (IR) spectroscopy was used to investigate the redox properties of recombinant alternative ubiquinol oxidase from Trypanosoma brucei, the organism responsible for African sleeping sickness. Stepwise reduction of the fully oxidized resting state of recombinant alternative ubiquinol oxidase revealed two distinct IR redox difference spectra. The first of these, signal 1, titrates in the reductive direction as an n = 2 Nernstian component with an apparent midpoint potential of 80 mV at pH 7.0. However, reoxidation of signal 1 in the same potential range under anaerobic conditions did not occur and only began with potentials in excess of 500 mV. Reoxidation by introduction of oxygen was also unsuccessful. Signal 1 contained clear features that can be assigned to protonation of at least one carboxylate group, further perturbations of carboxylic and histidine residues, bound ubiquinone, and a negative band at 1554 cm(-1) that might arise from a radical in the fully oxidized protein. A second distinct IR redox difference spectrum, signal 2, appeared more slowly once signal 1 had been reduced. This component could be reoxidized with potentials above 100 mV. In addition, when both signals 1 and 2 were reduced, introduction of oxygen caused rapid oxidation of both components. These data are interpreted in terms of the possible active site structure and mechanism of oxygen reduction to water.

FIGURE 1.

Reduced minus oxidized IR difference spectrum of rTAO. Reduction oxidation transformations were induced electrochemically in 100 mm potassium phosphate, 100 mm KCl at pH (black trace)/pD (red trace) 7.0 and containing 500 μm potassium ferricyanide and 100 μm benzyl viologen as redox mediators. Working electrode potentials were −400 mV for reduction and +550 mV for oxidation. For further details, see “Experimental Procedures.” Published ubiquinol-10 minus ubiquinone-10 redox spectra recorded in both H₂O (black) and D₂O (red) media are also displayed for qualitative comparisons (37).

FIGURE 2.

Reductive titration of rTAO. A, difference spectra recorded during a reductive titration in 100 mm potassium phosphate, 100 mm KCl at pH 7.0 (see “Experimental Procedures”). B, plots of peak/trough intensities at 1658/1641 and 1544/1554 cm⁻¹ versus potential. Data were simulated with a Nernst-derived equation for an n = 2 component with E_1/2 = 79 mV.

FIGURE 3.

Separation of redox spectra and oxidative titration of signal 1. A, a fully oxidized spectrum was recorded and use as a background. Potential was set to −10 mV, and signal 1 was recorded after 20 min. A new background spectrum was then recorded, and signal 2 was observed to develop at 1 h. B, oxidative titration of signal 1 at pH 7.0 with a mixture of redox mediators containing ferricyanide. Data were simulated with a Nernst-derived equation (see “Experimental Procedures”) for an n = 2 component with E_1/2 = 520 mV (solid line) or for an unrestricted fit which gave n = 0.6 and E_1/2 = 510 ± 30 mV (dotted line). The maximum intensity of signal 1 was determined by the addition of dioxygen to the fully reduced state.

FIGURE 4.

Summary of experimental observations and their relation to the catalytic cycle of reduction of oxygen to water.