A H₂O‐Producing NADH Oxidase from the Protozoan Parasite Giardia Duodenalis

Abstract

We describe the purification of a H,O-producing NADH oxidase from the protozoan parasite Giardiu duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1 : 1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K, value of 4.2 pM for NADH and 16 pM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce 0;-nor H,O, as stoichiometric products of oxygen reduction, implicating H,O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (CU'+ and ZnZ+ ) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P) ' oxidoreductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondria1 cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardiu close to the anaerobic bacteria in evolutionary terms.

Drug Targets and Mechanisms of Resistance in the Anaerobic Protozoa

“…Furazolidone resistance has also been induced in Giardia (22,195), and NADH oxidase has been shown to activate it to its free radical state (22,24). These lines were also more easily induced to quinacrine resistance, generating a second type of multidrug resistance (200).…”

“…There is no evidence for O 2 utilization in Giardia, although O 2 has been shown to be consumed (110,229). Alternatively, it has been shown that O 2 can be converted directly to H 2 O by NADH oxidase to protect the key anaerobic enzymes in the glycolytic pathway, PFOR and ferredoxin, for example (22,24). These components have been purified and their activity has been demonstrated in vitro (194,196).…”

“…No cytochromes have been found in Giardia. A terminal oxidase which converts oxygen directly to water scavenges oxygen from this microaerotolerant organism to protect the anaerobic PFOR and ferredoxins (24). This 46-kDa NADH oxidase is the only aerobic enzyme so far described in Giardia and is a homologue of the enzyme found in anaerobic bacteria.…”

The antioxidant potential of pyruvate in the amitochondriate diplomonads Giardia intestinalis and Hexamita inflata

“…An Fe-type superoxide dismutase (SOD) has been described for H. inflata (Biagini et al, 1997) and, although SOD activity was initially reported in Giardia (Thompson et al, 1993), in a more recent study SOD activity was undetectable even after induction with 1,10-phenanthroline (Brown et al, 1995). In addition, G. intestinalis contains a thioredoxin-reductase-like disulphide reductase which has the ability to reduce cystine (Brown et al, 1996), whereas H. inflata contains an as yet uncharacterized thiol reductase which may be involved in cystine reduction (Biagini et al, 1997).…”

NAD(P)H:menadione oxidoreductase of the amitochondriate eukaryote Giardia lamblia: a simpler homologue of the vertebrate enzyme The GenBank accession number for the glQR1-encoding sequence reported in this paper is AF321405.

“…Both species lack catalase, glutathione and glutathione reductase ; moreover no superoxide dismutase (SOD) has been detected in G. lamblia (Brown et al, 1995). Proposed components of alternative mechanisms of oxygen detoxification (oxidation defence) in G. lamblia include a cytosolic H # O-forming NADH oxidase, proposed to function as a terminal oxidase that removes excess reducing equivalents and as a component of the electron-transport pathway that protects oxygenlabile proteins by maintaining a reduced intracellular environment (Brown et al, 1995(Brown et al, , 1996a(Brown et al, , 1998 ; a membrane-associated NADH peroxidase associated with the observed H # O # removal by viable trophozoites (Brown et al, 1995) ; and a thioredoxin-like disulfide reductase with a role in disulfide redox management (Brown et al, 1996b(Brown et al, , 1998.…”