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

Abstract

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 μmol min-1 mg-1 in native membrane vesicles to 4.7 μmol 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 ∼17 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 ∼1.2 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 KH(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.