Biochemistry. chemotherapeutic targets.13 Although the ETC is generally well conserved across species, the first component (Complex I) of the ETC is DNA sequence divergent and relatively uncharacterized. The Complex I of eukaryotes is typically composed of a multi-subunit NADH:Ubiquinone oxidoreductase that oxidizes NADH in a rotenone-sensitive manner. In contrast, encodes an alternative single polypeptide non-proton pumping enzyme that is rotenone-insensitive. Alternative NADH dehydrogenase (NDH2) enzymes are flavoproteins that catalyze the transfer of electrons from NADH to ubiquinone (CoQ(pfNDH2) is usually a single polypeptide, approximately 52 kDa in size.14 In an effort to explore targets along the ETC in in order to thoroughly evaluate substrate and inhibitor specificity. DIF Full-length protein was expressed in-frame with a C-terminal 6xHis tag. The presence of detergent (0.5% Triton X-100) was critical for purification of active enzyme. The enzymatic activity of pfNDH2 was measured by chemical quantification of NAD+ Paradol using an assay adapted from Putt et al. in which addition of acetophenone base, followed by incubation at 100 C with formic acid, yields a product with strong fluorescence emission at 444 nm when excited at 372 nm (Supplemental Scheme 1).28 The pH dependence of the pfNDH2 reaction was assessed using this fluorescence-based assay at fixed concentrations of NADH and CoQ0 (0.1 mM for both substrates). pfNDH2 was maximally active at a pH range between 7.0 and 9.0 (Supplemental Fig. 1). Given these data, a HEPES buffer equilibrated to pH 8.0 Paradol was used in subsequent kinetics assays. Similar to other CoQsubstrates, only CoQ0 and CoQ1 showed detectable catalysis with pfNDH2. CoQ0 afforded the maximal catalytic rate; however, the in the absence of mitochondrial membrane association. The inorganic electron acceptor, DCIP, functioned with comparable efficiency to CoQ0, albeit with increased substrate specificity and lower catalytic rate. In comparison, the efficiency of menadione was significantly reduced due to an increase in the have been based on sequence and structural similarities to other redox enzymes.30 The biochemical relevance of these predictions, however, has not yet been exhibited. An N-terminal truncation product of pfNDH2 (ND214) was expressed and purified in order to determine if the conserved GxGxxG domains are necessary for catalytic activity. Truncated pfNDH2 showed significantly less activity compared to full-length product arguing that this N-terminal region of the enzyme is critical for full catalytic activity (data not shown). NDH2 has been implicated as an activator of the plant-derived antimalarial, artemisinin.31 A knockout screen using homozygous deletion strains showed that deletion of two NDH2 genes resulted in artemisinin resistance. Although expression of pfNDH2 in NDH2 knockout strains partially restored artemisinin sensitivity, and over-expression of the NDH2 genes was shown to increase artemisinin sensitivity, no biochemical link between pfNDH2 and artemisinin has yet been established.31 Therefore artemisinin was tested for either activation or inhibition of pfNDH2 activity. We found that arteminsin, even at high concentrations, did not perturb catalysis by pfNDH2 (Table 2). These results suggest that artemisinin may act via an indirect mechanism with NDH2 rather than by direct enzyme binding. Table 2 Inhibition of in vitro enzyme activity and in vivo parasite proliferation proliferationa,bparasite proliferation.14, 32 Dibenziodolium chloride (DPI) and diphenyliodonium chloride (IDP) have been reported to inhibit pfNDH2 activity in crude lysate fractions (IC50 = 0.24 0.03 and 5.99 0.36, respectively), and Paradol both show efficacy against whole parasite proliferation.26 It has been suggested that this antimalarial mechanisms of DPI, IDP, and HDQ may be attributed to the inhibition of pfNDH2 activity, however, doseCeffect profiles using purified recombinant pfNDH2 did not corroborate these findings. In fact, these compounds did not inhibit pfNDH2 activity at concentrations of up to 10 M (Table 2). Both DPI and IDP are well-known flavoprotein oxidoreductase inhibitors, suggesting that previous observations of reduction in NADH consumption using crude parasite lysate may have been due to inhibition of a different flavoenzyme-dependent reaction.33C38 The chemical structure of HDQ is similar to that of the CoQsubstrate (Fig. 1) and thus, we speculated that its mode of action might be related to another CoQType II dihydroorotate dehydrogenase (pfDHOD). pfDHOD is a mitochondrial flavoenzyme that catalyzes the oxidation of dihydroorotate (l-DHO) using a FMN cofactor that is re-oxidized by CoQstrain expressing a Type I cytoplasmic DHOD from (scDHOD) was used to pinpoint the antimalarial mechanism of HDQ (Supplemental methods). Painter et al. had previously shown that this role of the mitochondrial electron potential in the asexual stage of growth was to maintain a pool of CoQin order.