Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase

Tuz, Karina; Li, Chen; Fang, Xuan; Raba, Daniel A.; Liang, Pingdong; Minh, David D. L.; Juárez, Óscar

doi:10.1074/jbc.m116.770982

Cited by 11 publications

(33 citation statements)

References 50 publications

(153 reference statements)

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“…It should be noted that the effects of HQNO were smaller compared to monensin, which may be due to a relatively low concentration of HQNO reaching C. trachomatis cells, due to the presence of albumin, or because both EBs and RBs are enclosed in an inclusion, which might decrease the permeability of this inhibitor. Alternatively, HQNO might not inhibit C. trachomatis Na ϩ -NQR as effectively as V. cholerae Na ϩ -NQR, considering that the ubiquinone-binding site (where HQNO is bound) has a 70% identity between these two bacteria (27). Also, the function of an Na ϩ /H ϩ antiporter (NhaD) (74) might maintain, to a certain extent, the sodium gradient across the C. trachomatis membrane (24,31,93).…”

Section: Effects Of Mitochondrial Inhibitors and Uncouplersmentioning

confidence: 99%

“…The chlamydial oxidative phosphorylation system consists of the sodium-dependent NADH dehydrogenase (Na ϩ -NQR), succinate dehydrogenase, cytochrome bd oxidase, and an A 1 -A 0 -ATPase (12,24,25). Furthermore, C. trachomatis seems to use menaquinone (26), instead of ubiquinone, which is used by mitochondria (27). Na ϩ -NQR is a unique respiratory complex that is analogous to the mitochondrial complex I, incorporating the electrons from NADH into ubiquinone, feeding the lower part of the respiratory chain (28).…”

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confidence: 99%

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Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation

Liang

Rosas‐Lemus²,

Patel

et al. 2018

Journal of Biological Chemistry

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is an obligate intracellular human pathogen responsible for the most prevalent sexually-transmitted infection in the world. For decades has been considered an "energy parasite" that relies entirely on the uptake of ATP from the host cell. The genomic data suggest that respiratory chain could produce a sodium gradient that may sustain the energetic demands required for its rapid multiplication. However, this mechanism awaits experimental confirmation. Moreover, the relationship of chlamydiae with the host cell, in particular its energy dependence, is not well understood. In this work, we are showing that has an active respiratory metabolism that seems to be coupled to the sodium-dependent synthesis of ATP. Moreover, our results show that the inhibition of mitochondrial ATP synthesis at an early stage decreases the rate of infection and the chlamydial inclusion size. In contrast, the inhibition of the chlamydial respiratory chain at mid-stage of the infection cycle decreases the inclusion size but has no effect on infection rate. Remarkably, the addition of monensin, a Na/H exchanger, completely halts the infection. Altogether, our data indicate that chlamydial development has a dynamic relationship with the mitochondrial metabolism of the host, in which the bacterium mostly depends on host ATP synthesis at an early stage, and at later stages it can sustain its own energy needs through the formation of a sodium gradient.

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Section: Effects Of Mitochondrial Inhibitors and Uncouplersmentioning

confidence: 99%

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confidence: 99%

Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation

Liang

Rosas‐Lemus²,

Patel

et al. 2018

Journal of Biological Chemistry

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“…The predicted site is not only inconsistent with the location of the putative cavity in NqrA (13), but is also too far from the riboflavin (ϳ36 Å) for efficient electron transfer. Because the work by Tuz et al (14) was largely based on steadystate kinetic analysis of mutants, it is difficult to determine whether the critical residues that they identified (NqrB-Phe-211 and -Phe-213) are directly involved in forming the binding pocket for the ubiquinone ring or whether they affect the reaction of ubiquinone indirectly though some long-range conformational change. In addition, 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 Na ϩ -NQR (10,14).…”

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confidence: 99%

“…Because the work by Tuz et al (14) was largely based on steadystate kinetic analysis of mutants, it is difficult to determine whether the critical residues that they identified (NqrB-Phe-211 and -Phe-213) are directly involved in forming the binding pocket for the ubiquinone ring or whether they affect the reaction of ubiquinone indirectly though some long-range conformational change. In addition, 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 Na ϩ -NQR (10,14). However, interpretation of data obtained through this method can be problematic because it may be impossible to distinguish between specific and non-specific binding of the hydrophobic ligands to the enzyme, particularly in the case of an integral membrane protein.…”

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confidence: 99%

“…Recently, by alanine-scanning mutagenesis of aromatic residues in NqrB and molecular docking, Tuz et al (14) proposed that the binding site of the ubiquinone ring is located at the interface of the NqrB and NqrD subunits. The predicted site is not only inconsistent with the location of the putative cavity in NqrA (13), but is also too far from the riboflavin (ϳ36 Å) for efficient electron transfer.…”

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Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

Ito

Murai

Ninokura

et al. 2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeThe 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. I]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.The Na ϩ -pumping NADH-quinone oxidoreductase (Na ϩ -NQR) 2 is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, such as Vibrio cholerae and Haemophilus influenzae (1, 2). Na ϩ -NQR obtains energy by oxidizing NADH and reducing ubiquinone, which allows it to generate an electrochemical Na ϩ gradient across the inner bacterial membrane. The enzyme is an integral membrane complex consisting of six subunits (NqrA-F) encoded by the nqr operon (2). There is a consensus that the electron transfer takes place through a series of five redox cofactors as follows: a FAD; a 2Fe-2S center; two covalently bound FMN, and a riboflavin (3-5). Different studies have intensively investigated the locations and redox properties of the cofactors of the enzyme (6 -12); however, the exact locations of the ubiquinone-binding site(s) and the Na ϩ transport pathway, as well as the mechanism that couples Na ϩ transport to the electron transfer, still remain elusive.A recently published X-ray crystallographic study provided important information about the structure of V. cholerae Na ϩ -NQR (13), but the model includes several features that are difficult to reconcile with previous biochemical and/or biophysical functional characterizations (11). For instance, the spatial distances between several pairs of redox cofactors in the proposed electron transfer pathway are too large to support physiological rates of electron transfer; for example, the edge-toedge distance between the [2Fe-2S] cluster in NqrF and the Fe(Cys) 4 in NqrD (33.4 Å) and between the FMN and the riboflavin cofactor in NqrB (29.3 Å). The fact that electron transfer between these cofactors takes place indicates that the subunits harboring the cofactors undergo large conformational changes during turnover that decrease these spatial gaps (13).Additionally, the crystallographic model lacks an anticipated tightly bound quinone, which has been reported in the enzyme preparations from different laboratories (4,6,8) and suggested to be located in NqrA on the basis of photoaffinity labeling and NMR studies (7,9). In the crystallographic model, the NqrA subunit includes a deep cavity that is large enough to accommodate a ubiquinone molecule, but the cavity is ϳ20 Å above the predicted membrane surface and the distance between the cavity and the riboflavin in NqrB subunit is too large (Ͼ40 Å) to be consistent with electron transfer during tu...

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Electrostatics and water occlusion regulate covalently‐bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR

et al. 2021

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Proteins like NADH:ubiquinone oxidoreductase (NQR), an essential enzyme and ion pump in the physiology of several pathogenic bacteria, tightly regulate the redox properties of their cofactors. Although flavin mononucleotide (FMN) is fully reduced in aqueous solution, FMN in subunits B and C of NQR exclusively undergo one‐electron transitions during its catalytic cycle. Here, we perform ab initio calculations and molecular dynamics simulations to elucidate the mechanisms that regulate the redox state of FMN in NQR. QM/MM calculations show that binding site electrostatics disfavor anionic forms of FMNH2, but permit a neutral form of the fully reduced flavin. The potential energy surface is unaffected by covalent bonding between FMN and threonine. Molecular dynamics simulations show that the FMN binding sites are inaccessible by water, suggesting that further reductions of the cofactors are limited or prohibited by the availability of water and other proton donors. These findings provide a deeper understanding of the mechanisms used by NQR to regulate electron transfer through the cofactors and perform its physiologic role. They also provide the first, to our knowledge, evidence of the simple concept that proteins regulate flavin redox states via water occlusion.

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Identification of the Catalytic Ubiquinone-binding Site of Vibrio cholerae Sodium-dependent NADH Dehydrogenase

Cited by 11 publications

References 50 publications

Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation

Dynamic energy dependency of Chlamydia trachomatis on host cell metabolism during intracellular growth: Role of sodium-based energetics in chlamydial ATP generation

Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

Electrostatics and water occlusion regulate covalently‐bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR

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