Abstract
Type II NADH:quinone oxidoreductase (NDH-2) plays a crucial role in the respiratory chains of many organisms. Its absence in mammalian cells makes NDH-2 an attractive new target for developing antimicrobials and antiprotozoal agents. We established a novel bioelectrochemical platform to characterize the catalytic behavior of NDH-2 from Caldalkalibacillus thermarum and Listeria monocytogenes strain EGD-e while bound to native-like lipid membranes. Catalysis of both NADH oxidation and lipophilic quinone reduction by membrane-bound NDH-2 followed the Michaelis-Menten model; however, the maximum turnover was only achieved when a high concentration of quinone (>3 mM) was present in the membrane, suggesting that quinone availability regulates NADH-coupled respiration activity. The quinone analogue 2-heptyl-4-hydroxyquinoline-N-oxide inhibited C. thermarum NDH-2 activity, and its potency is higher in a membrane environment compared to assays performed with water-soluble quinone analogues, demonstrating the importance of testing compounds under physiologically relevant conditions. Furthermore, when phenothiazines, one of the most commonly identified NDH-2 inhibitors, were tested, they did not inhibit membrane-bound NDH-2. Instead, our assay platform unexpectedly suggests a novel mode of phenothiazine action where chlorpromazine, a promising antitubercular agent and key medicine used to treat psychotic disorders, is able to disrupt pH gradients across bacterial membranes.
Highlights
NADH dehydrogenase/NADH:quinone oxidoreductase (NDH) is a key respiratory enzyme in many organisms
Impedance spectroscopy confirmed that planar membranes containing C. thermarum NDH-2 spontaneously formed after applying a solution of Escherichia coli polar lipid proteoliposomes, premixed with purified C. thermarum NDH2, onto an electrode with a self-assembled monolayer containing cholesterol “tethers” (Figure 1B).[35]
We did not observe unusual catalytic behaviors such as an allosteric mechanism as proposed for P. falciparum NDH-232 or the two quinone-binding model that was proposed by Godoy-Hernandez et al.[39]
Summary
NADH dehydrogenase/NADH:quinone oxidoreductase (NDH) is a key respiratory enzyme in many organisms This class of enzymes catalyzes oxidation of NADH and reduction of quinone and plays a crucial role in maintaining the cellular NAD+/NADH balance and serves as a primary electron entry point for the respiratory chain to drive ATP synthesis. This might be advantageous for some organisms in the maintenance of the NAD+/NADH redox balance and generation of ATP because its catalytic function cannot be compromised by proton motive force back-pressure
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