Role of mitochondria in the leishmanicidal effects and toxicity of acyl phloroglucinol derivatives: nemorosone and guttiferone A

SUMMARYNemorosone (Nem) and guttiferone A (GutA) are acyl phloroglucinol derivatives (APD) that are present in different natural products. For both compounds anti-cancer and anti-microbial properties have been reported. In particular, an anti-leishmanial activity of both compounds was demonstrated. The aim of this study was to explore the possible role of mitochondria in the anti-leishmanial activity of Nem and GutA in comparison with their action on mammalian mitochondria. Both APD inhibited the growth of promastigotes ofLeishmania tarentolae(LtP) with half maximal inhibitory concentration (IC50) values of 0·67 ± 0·17 and 6·2 ± 2·6μm; while IC50values for cytotoxicity against peritoneal macrophages from BALB/c mice were of 29·5 ± 3·7 and 9·2 ± 0·9μm, respectively. Nemorosone strongly inhibited LtP oxygen consumption, caused species-specific inhibition (P< 0·05) of succinate:ubiquinone oxidoreductase (complex II) from LtP-mitochondria and significantly increased (P< 0·05) the mitochondrial superoxide production. In contrast, GutA caused only a moderate reduction of respiration in LtP and triggered less superoxide radical production in LtP compared with Nem. In addition, GutA inhibited mitochondrial complex III in bovine heart submitochondrial particles, which is possibly involved in its mammalian toxicity. Both compounds demonstrated at low micromolar concentrations an effect on the mitochondrial membrane potential in LtP. The present study suggests that Nem caused its anti-leishmanial action due to specific inhibition of complexes II/III of mitochondrial respiratory chain ofLeishmaniaparasites that could be responsible for increased production of reactive oxygen species that triggers parasite death.

The Quinone Binding Site in Escherichia coli Succinate Dehydrogenase Is Required for Electron Transfer to the Heme b

We have examined the role of the quinone-binding (QP) site of Escherichia coli succinate:ubiquinone oxidoreductase (succinate dehydrogenase) in heme reduction and reoxidation during enzyme turnover. The SdhCDAB electron transfer pathway leads from a cytosolically localized flavin adenine dinucleotide cofactor to a QP site located within the membrane-intrinsic domain of the enzyme. The QP site is sandwiched between the [3Fe-4S] cluster of the SdhB subunit and the heme b556 that is coordinated by His residues from the SdhC and SdhD subunits. The intercenter distances between the cluster, heme, and QP site are all within the theoretical 14 Å limit proposed for kinetically competent intercenter electron transfer. Using EPR spectroscopy, we have demonstrated that the QP site of SdhCDAB stabilized a ubisemiquinone radical intermediate during enzyme turnover. Potentiometric titrations indicate that this species has an Em,8 of ∼60 mV and a stability constant (KSTAB) of ∼1.0. Mutants of the following conserved QP site residues, SdhC-S27, SdhC-R31, and SdhD-D82, have severe consequences on enzyme function. Mutation of the conserved SdhD-Y83 suggested to hydrogen bond to the ubiquinone cofactor had a less severe but still significant effect on function. In addition to loss of overall catalysis, these mutants also affect the rate of succinate-dependent heme reduction, indicating that the QP site is an essential stepping stone on the electron transfer pathway from the [3Fe-4S] cluster to the heme. Furthermore, the mutations result in the elimination of EPR-visible ubisemiquinone during potentiometric titrations. Overall, these results demonstrate the importance of a functional, semiquinone-stabilizing QP site for the observation of rapid succinate-dependent heme reduction.