b. brucei cells (strain Lister 427, cell line 449) were cultured up to the exponential growth phase and homogenates were obtained by grinding the washed parasite pellets with silicon-carbide abrasive grain (mesh 300) in disruption such buffer containing 250 mM sucrose, 25 mM Tris-HCl and l mM EDTA, pH 7.8 (STE buffer). Differential centrifugation [33] was performed as follows: the suspension was taken up in another 3 ml STE buffer and centrifuged at 30 g for 3 min. The supernatant, representing the cell homogenate, was centrifuged at 1,500 g for 10 min giving the nuclear fraction. The post-nuclear supernatant was then centrifuged at 5,000 g for 10 min giving the large-granular (mitochondria-enriched) fraction as pellet. This fraction was resuspended in 300 ��l STE buffer.

Oxygen consumption measurements Oxygen consumption was monitored in a thermostated vessel at 37��C with a Clark electrode (oxygraph). Measurements in permeabilized bloodstream-form T. brucei were done in buffer 1 (96.9 mM NaCl, 3.1 mM KCl, 5 mM MgCl2, 2 mM Na2HPO4, 90 mM Tris at pH 7.5). Batches of 2��107 cells were pelleted, washed in buffer 1 and incubated for 5�� on ice in 1 ml of buffer 1 containing 1 ng digitonin to permeabilize the cells. Subsequently the permeabilized cells were pelleted and washed twice with buffer 1 without digitonin after which the pellet was taken up in 1 ml buffer 1 and transferred to the oxygraph. Substrates and inhibitors were added as described in the text. 2 mM salicylhydroxamic acid (SHAM) was used to completely inhibit the T. b. brucei alternative oxidase (TAO) that is part of the GPO.

B6 and SHAM were dissolved in DMSO. The DMSO concentration used in these oxygen consumption measurements was less than 0.7%. Measurement of hydrogen peroxide production The method used to measure H2O2 production in T. b. brucei mitochondria is based on the fluorogenic probe 2��,7��-dichlorodihydrofluorescein diacetate (H2DCFDA) which emits an intense green fluorescence only after deacetylation and subsequent oxidation. ROS production by the mitochondrial fraction of bloodstream form T. b. brucei was measured in a 96-well microtiter plate using a fluorescence plate reader (Victor Wallace multiplate reader). In each well, 0.25 mg mitochondrial protein/ml and 5 ��M H2DCFDA to a final volume of 0.2 ml with 10 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, pH 7.5 were present.

The reaction, performed at 25��C, was started by the addition of 10 mM G3P, in the presence and absence of 10 ��M B6. T. b. brucei growth conditions We used bloodstream and procyclic forms of T. b. brucei strain Lister 427, cell lines AV-951 449 [34], constitutively expressing the E. coli tetracycline (Tet) repressor gene via the chromosomally integrated plasmid pHD449 that also confers phleomycin resistance. Bloodstream forms were cultured in HMI-9 medium containing 10% heat-inactivated foetal calf serum (Invitrogen) and 0.