Type II
NADH:
quinone oxidoreductases (ndh) are flavoenzymes found in a broad range of organisms including plants, fungi, protozoa, and bacteria. The ndh
enzymes catalyze the oxidation of
NADH with concomitant reduction of
quinone (Q). These membrane-bound respiratory
enzymes differ from the canonical
NADH: dehydrogenase (complex I), because they are not involved in the vectorial transfer of
protons across membranes. In Plasmodium falciparum and Mycobacterium tuberculosis, causative agents of
malaria and
tuberculosis, respectively,
ndhs have aroused interest because of the essential role played in maintaining a reduced Q-pool during
infection. In this chapter, we present methods for the measurement of steady-state parameters for
ndhs from both pathogens, highlighting best practices and caveats. In addition, owing to the interest in
ndhs as potential chemotherapeutic targets, we describe a miniaturized endpoint assay that is validated for high-throughput screening (HTS) of
chemical libraries.