Redox-Dependent Sodium Binding by the Na<sup>+</sup>-Translocating NADH:Quinone Oxidoreductase from <i>Vibrio harveyi</i>

Bogachev, Alexander V.; Bertsova, Yulia V.; Aitio, Olli; Permi, Perttu; Verkhovsky, Michael I.

doi:10.1021/bi700440w

Cited by 23 publications

(8 citation statements)

References 27 publications

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“…In contrast with the conclusions of the earlier 23 Na NMR study (36), our results clearly show that the affinity of Na ϩ -NQR for sodium does not change between the fully oxidized and fully reduced states of the enzyme.…”

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confidence: 99%

“…To determine both the number of cation-binding sites and their affinity, equilibrium 22 Na binding experiments were performed in the oxidized and fully reduced forms of the enzyme. Previously, Bogachev et al (38) studied the interactions of sodium with Vibrio harveyi Na ϩ -NQR, using 23 Na NMR spectroscopy (36). They found that the line width of the 23 Na NMR signal was significantly larger in the presence of the reduced, than the oxidized, enzyme and calculated sodium affinity constants of 30 M and 24 mM, respectively.…”

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confidence: 99%

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The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Juárez

Shea

Makhatadze

et al. 2011

Journal of Biological Chemistry

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The Na ؉ -translocating NADH:quinone oxidoreductase is the entry site for electrons into the respiratory chain and the main sodium pump in Vibrio cholerae and many other pathogenic bacteria. In this work, we have employed steady-state and transient kinetics, together with equilibrium binding measurements to define the number of cation-binding sites and characterize their roles in the enzyme. Our results show that sodium and lithium ions stimulate enzyme activity, and that Na ؉ -NQR enables pumping of Li ؉ , as well as Na ؉ across the membrane.We also confirm that the enzyme is not able to translocate other monovalent cations, such as potassium or rubidium. Although potassium is not used as a substrate, Na ؉ -NQR contains a regulatory site for this ion, which acts as a nonessential activator, increasing the activity and affinity for sodium. Rubidium can bind to the same site as potassium, but instead of being activated, enzyme turnover is inhibited. Activity measurements in the presence of both sodium and lithium indicate that the enzyme contains at least two functional sodium-binding sites. We also show that the binding sites are not exclusively responsible for ion selectivity, and other steps downstream in the mechanism also play a role. Finally, equilibrium-binding measurements with 22 Na ؉ show that, in both its oxidized and reduced states, Na ؉ -NQR binds three sodium ions, and that the affinity for sodium is the same for both of these states.The sodium-pumping NADH:quinone oxidoreductase (Na ϩ -NQR) 2 is the entry site for electrons into the respiratory chain of many marine and pathogenic bacteria, including Vibrio cholerae (1-9). Na ϩ -NQR catalyzes the electron transfer from NADH to ubiquinone, and uses the energy released by the redox reaction to pump sodium ions, creating an electrochemical gradient (10 -13). In different bacteria, the sodium gradient supplies energy for a variety of fundamental processes, including amino acid and sugar transport, ATP synthesis, pH regulation, drug efflux, and toxin extrusion (12). Na ϩ -NQR is composed of five subunits with transmembrane spanning segments (NqrB-F) and one hydrophilic subunit (NqrA) (14). The enzyme contains five redox cofactors: two covalently bound FMNs, in the hydrophilic domains of subunits NqrB (FMN B ) and NqrC (FMN C ) (15-19), one FAD and one 2Fe-2S center, in the cytosolic domain of NqrF (20, 21), and one riboflavin in . With the exception of riboflavin, all of the cofactor-binding sites have been identified in the sequence. Flavin molecules are flexible electron carriers that can participate in one-or two-electron reactions, although the two-electron reactions are thermodynamically favored and produce relatively more stable products. Interestingly, in Na ϩ -NQR, the two FMNs and the riboflavin cofactor undergo only one-electron transitions in the reaction cycle of the enzyme. Furthermore, riboflavin is found as a stable neutral radical (RibH ⅐ ) in the air-oxidized form of the enzyme (15).Our group has previously shown that electrons move thro...

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confidence: 99%

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Juárez

Shea

Makhatadze

et al. 2011

Journal of Biological Chemistry

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“…Thus, dependence of the rate of the ( L↔M ) → O transition on Na + concentration can be used to determine the kinetic binding constant of this ion as ≈ 4.7×10 3 M –1 s –1 . Typically, the rate constant of Na + binding with macromolecules is in the range 10 7 – 10 9 M –1 s –1 26 27 28 . Thus, Na + diffuses to its final binding site in NaR at least four orders of magnitude slower than in most other sodium-binding proteins.…”

Section: Discussionmentioning

confidence: 99%

Real-time kinetics of electrogenic Na+ transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95

Bogachev

Bertsova

Verkhovskaya

et al. 2016

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Discovery of the light-driven sodium-motive pump Na+-rhodopsin (NaR) has initiated studies of the molecular mechanism of this novel membrane-linked energy transducer. In this paper, we investigated the photocycle of NaR from the marine flavobacterium Dokdonia sp. PRO95 and identified electrogenic and Na+-dependent steps of this cycle. We found that the NaR photocycle is composed of at least four steps: NaR519 + hv → K585 → (L450↔M495) → O585 → NaR519. The third step is the only step that depends on the Na+ concentration inside right-side-out NaR-containing proteoliposomes, indicating that this step is coupled with Na+ binding to NaR. For steps 2, 3, and 4, the values of the rate constants are 4×104 s–1, 4.7 × 103 M–1 s–1, and 150 s–1, respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Δψ ~ 200 mV) generated by a single turnover of NaR incorporated into liposomes and attached to phospholipid-impregnated collodion film. On the basis of these observations, a mechanism of light-driven Na+ pumping by NaR is suggested.

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“…With this assignment, we can begin to investigate the role of riboflavin in the mechanism of the enzyme. Bogachev et al (21,24,25) observed the disappearance of a neutral flavosemiquinone species during the reduction of the enzyme by NADH, in optically monitored stopped flow measurements. This indicates that riboflavin plays a role in the redox mechanism of Na ϩ -NQR.…”

Section: Hplc Identification Of the Soluble Flavins In The Double Mutmentioning

confidence: 99%

Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

Juárez

Nilges

Gillespie

et al. 2008

Journal of Biological Chemistry

103

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Here we present new evidence that riboflavin is present as one of four flavins in Na ؉ -NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na ؉ -NQR.The Na ϩ

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Redox-Dependent Sodium Binding by the Na⁺-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

Cited by 23 publications

References 27 publications

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Real-time kinetics of electrogenic Na+ transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95

Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

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Redox-Dependent Sodium Binding by the Na+-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

Cited by 23 publications

References 27 publications

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae

Real-time kinetics of electrogenic Na+ transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95

Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

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Redox-Dependent Sodium Binding by the Na⁺-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi