Specific Modification of a Na + Binding Site in NADH:Quinone Oxidoreductase from Klebsiella pneumoniae with Dicyclohexylcarbodiimide

Abstract

ABSTRACT The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that translocates protons (or in some cases Na + ) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the Na + -translocating complex I from the enterobacterium Klebsiella pneumoniae with N , N ′-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for Na + binding. At low Na + concentrations (0.6 mM), DCCD inhibited both quinone reduction and Na + transport by NDH-1 concurrent with the covalent modification of a 30-kDa polypeptide. In the presence of 50 mM Na + , NDH-1 was protected from inhibition by DCCD, and the modification of the 30-kDa polypeptide with [ 14 C]DCCD was prevented, indicating that Na + and DCCD competed for the binding to a critical carboxyl group in NDH-1. The 30-kDa polypeptide was assigned to NuoH, the homologue of the ND1 subunit from mitochondrial complex I. It is proposed that Na + binds to the NuoH subunit during NADH-driven Na + transport by NDH-1.