Characterization of the respiratory chain of Leishmania donovani promastigotes

Abstract

Inhibition analysis of respiration of Leishmania donovani promastigotes in resting, starved and permeabilized cells in the presence of classical electron transfer complex inhibitors such as rotenone, thenoyltrifluoroacetone and antimycin demonstrated the absence of complex I component of the respiratory chain in this organism. Cyanide failed to completely block the oxygen uptake (residual 25-30%) even at high concentrations. The alternative oxidase inhibitor for Trypanosoma brucei, salicylhydroxamic acid (SHAM) had no effect on respiration while the cytochrome o inhibitor orthohydroxydiphenyl (OHD) could block cyanide-insensitive respiration at low concentrations. Succinate-dependent O₂ uptake in permeabilized cells follows the classical pathway. Oxidation of NADH by a membrane-rich fraction produced H₂O₂ as the end product and was insensitive to repiratory chain inhibitors. The presence of NADH-fumarate reductase was demonstrated in membrane-rich fraction and fumarate could reduce H₂O₂ production from NADH indicating fumarate to be an endogenous substrate for accepting electrovs from NADH. A differential route for NADH oxidation was further confirmed by NADH cytochrome c reductase insensitivity to antimycin. A tentative scheme for electron transfer pathway in this organism is proposed in which a reversal of Krebs cycle enzymes occur producing succinate that can be excreted or oxidized depending upon the energy demands of the cell. Inhibition studies also suggest bifurcations of the respiratory chain that can be of minor importance for the organism.