A H2O-PRODUCING NADH OXIDASE FROM THE PROTOZOAN PARASITE GIARDIA-DUODENALIS

We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavo protein containing flavin adenine dinucleotide in a 1:1 molar ratio wi th the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophor esis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K-m value of 4.2 mu M for NADH and 16 mu M for NADPH (pH 7.8 at room temperature). With oxygen as the prima ry electron acceptor under aerobic conditions, the pure enzyme did not produce O-2(.-) nor H2O2 as stoichiometric products of oxygen reducti on, implicating H2O as the end product and obviating the need for supe roxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibite d this activity. Under anaerobic conditions the enzyme catalysed elect ron transfer at lower efficiencies to other electron accepters includi ng nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using e ither NADH or NADPH as electron donors. NADPH, however, was a more eff icient electron donor. Cytochrome c was not reduced under any assay co nditions used. The enzyme reduced the nitrofuran drugs, furazolidone ( an antigiardial) and nitrofurantoin, to their toxic radical forms as d etermined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pu re NADH oxidase did not demonstrate ferredoxin:NAD(P)(+) oxido-reducta se activity since it could not accept electrons from reduced ferredoxi n to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore , function as a terminal oxidase, similar to the mitochondrial cytochr ome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.