Specific modification of a Na+ binding site in NADH:quinone oxidoreductase from Klebsiella pneumoniae with dicyclohexylcarbodiimide

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 [14C]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.

FIG. 1.

Identification of proteins in NDH-1 and modification of subunit NuoH with [¹⁴C]DCCD. Panel A shows the SDS-PAGE of NDH-1 (100 μg) after Source 15Q chromatography (lane 1) and the 2-butanol extract from 2 mg NDH-1 (lane 2). The gels were stained with Coomassie. Polypeptides were identified by mass spectroscopy (see Table 1 for results) or N-terminal sequencing (indicated with an asterisk). Nuo and Atp denote subunits of NDH-1 and the F₁F_o ATPase, respectively. Panel B shows the autoradiograms of NDH-1 after modification with [¹⁴C]DCCD and separation by SDS-PAGE. Two aliquots of NDH-1 (30 μg each) were mixed with [¹⁴C]DCCD in the presence of 0.6 mM Na⁺ (lane 3) or 50 mM Na⁺ (lane 4). Lane 5 shows the autoradiogram of the 2-butanol extract from 2 mg NDH-1 after modification with [¹⁴C]DCCD in the presence of 0.6 mM Na⁺.

FIG. 2.

Influence of pH on NADH-driven Na⁺ transport by NDH-1. NADH-driven Na⁺ uptake (•) by NDH-1 purified by anionic-exchange chromatography in the buffer used for the reconstitution of the complex into proteoliposomes (10 mM HEPES-KOH, 50 mM KCl, 10% glycerol) was monitored. The protein-to-lipid ratio was 1:4. At time zero, 0.1 mM NADH and 5 mM NaCl were added, and the Na⁺ content of the proteoliposomes was determined at the indicated times. In the control reactions, NADH was omitted (▪). (A) Proteoliposomes containing NDH-1. Na⁺ transport at pH 6.5 or pH 7.5 was monitored. (B) Proteoliposomes containing NDH-1 and the H⁺-translocating ATPase. Na⁺ transport at pH 7.0 was monitored.

FIG. 3.

Proton transport into proteoliposomes containing NDH-1 from Klebsiella pneumoniae and the F₁F_o ATPase from Escherichia coli. Proton transport was followed by the quenching of ACMA fluorescence in 10 mM Tris-HCl, pH 7.0, containing 10 mM MgCl₂, 50 mM KCl, 10% glycerol, and 5 mM NaCl. The arrows indicate the addition of 0.1 mM NADH or 2.5 mM ATP.

FIG. 4.

pH optimum for the modification of NDH-1 by DCCD. NDH-1 obtained by PEG precipitation was adjusted to the desired pH with Good buffers and incubated with 698 μM DCCD for 3 min. The control mixtures contained 6 μl ethanol. The residual Na⁺ concentration was 0.6 mM. The Q reductase activity of NDH-1 modified with DCCD (▾) or without DCCD added (▪) was determined at pH 8.0.

FIG. 5.

DCCD inhibition of quinone reduction by NDH-1. Aliquots of NDH-1 obtained by PEG precipitation (0.3 mg in 50 μl 50 mM HEPES-KOH, pH 7.5, 50 mM KCl, and 4% glycerol) were incubated with 0, 100, 200, or 500 μM DCCD for 4 min, and the quinone reductase activity was monitored at pH 8.0.

FIG. 6.

DCCD inhibition of Na⁺ transport by NDH-1. NDH-1 obtained by PEG precipitation was reconstituted into proteoliposomes at pH 7.5 and incubated with 200 μM (○), 500 μM (▪), or 800 μM (▾) DCCD or without DCCD (•). The protein-to-lipid ratio was 1:1. Na⁺ transport was initiated by the addition of 5 mM NaCl and 0.1 mM NADH. The data were corrected by subtracting the Na⁺ content of proteoliposomes from a control reaction without NADH added (40 to 80 nmol Na⁺ mg⁻¹ lipid) (Fig. 2).

FIG. 7.

Protection of NDH-1 from DCCD inhibition by Na⁺. NDH-1 obtained by PEG precipitation (0.14 mg in 20 μl 1 M MES, pH 6.5) was mixed with 698 μM DCCD, and the quinone reduction activity was monitored after different reaction times (final DCCD concentration in the assay buffer, 14 μM). The arrow indicates the addition of DCCD at time zero. ○, with DCCD added; •, without DCCD added; ▾, 50 mM NaCl added prior to DCCD addition.