The respiratory complex I (NDH I) from Klebsiella pneumoniae, a sodium pump

The electrogenic NADH:Q oxidoreductase from the enterobacterium Klebsiella pneumoniae transports Na+ ions. The complex was purified with an increase of the specific Na+ transport activity from 0.2 mumol min(-1) mg(-1) in native membrane vesicles to 4.7 mumol min(-1) mg(-1) in reconstituted enzyme specimens. The subunit pattern resembled that of complex I from Escherichia coli, and two prominent polypeptides were identified as the NuoF and NuoG subunits of complex I. During purification the typical cofactors of complex I were enriched to yield similar to17 nmol mg(-1) iron, 24 nmol mg(-1) acid-labile sulfide, and 0.79 nmol mg(-1) FMN in the purified sample. The enzyme contained similar to1.2 nmol mg(-1) Q6 and 1.5 nmol mg(-1) Q8. The reduction of ubiquinone by NADH was Na+-dependent, which indicates coupling of the chemical and the vectorial reaction of the pump. The Na+ activation profile corresponded to the Hill equation with a Hill coefficient K-H(Na+) = 1.96 and with a half-maximal saturation at 0.33 mm Na+. The reconstituted complex I from Klebsiella pneumoniae catalyzed deamino-NADH oxidation, Q1 reduction, and Na+ translocation with specific activities of 2.6 units mg(-1), 2.4 units mg(-1), and 4.7 units mg(-1), respectively, which indicate a Na+/electron stoichiometry of one.