Abstract
The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32-Met-39 and Phe-131-Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43-Lys-54 and Trp-23-Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.
Highlights
The Na؉-pumping NADH-quinone oxidoreductase (Na؉NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae
To more precisely locate the binding sites for ubiquinone and inhibitors, we have carried out photoaffinity labeling experiments with a photoreactive ubiquinone (PUQ-3) and two different aurachin-type inhibitors ([125I]PAD-1 and [125I]PAD-2) using the isolated Naϩ-NQR
The photoaffinity labeling experiment using PUQ-3 showed that the ubiquinone ring binds primarily in the region Ile-2– Met-39 of NqrA and secondarily in the region Val-94 –Met-154 of the same subunit (Fig. 4)
Summary
The Na؉-pumping NADH-quinone oxidoreductase (Na؉NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43–Lys-54 and Trp-23–Gly-89, respectively These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. We found that photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na؉-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work. To determine the number of binding sites and/or the dissociation constant for short-chain ubiquinone and the inhibitor HQNO, previous studies employed the equilibrium dialysis method with isolated wild-type and mutated Naϩ-NQR [10, 14]. In an attempt to explain these apparently paradoxical results, we propose models for the catalytic reaction of Naϩ-NQR and the interaction with inhibitor on the basis of the biochemical and biophysical information obtained in this study and previous works
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