Redox potentials of active-site bis(cysteinyl) fragments of thiol-protein oxidoreductases

Siedler, Frank; Rudolph‐Böhner, Sabine; Doi, Masao; Hj, Musiol; Moröder, Luis

doi:10.1021/bi00080a021

Cited by 77 publications

(60 citation statements)

References 40 publications

(33 reference statements)

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“…Indeed, the disruption of the bridge by mutagenesis modified notably Nox4 ROS production [43]. Disulfide bridges with a standard redox potential (E 0 ) of À180 mV [47] could be reduced by NADPH, NADH and b-mercaptoethanol that possess a lower E 0 (À324, À320, and À253 mV, respectively) [48]. Our results using those reducing agents indicated that Nox4 activity could be stimulated by extracellular redox modifications, possibly through the reduction of its disulfide bridge.…”

Section: Discussionmentioning

confidence: 76%

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

Nguyen

Lardy

Rousset

et al. 2013

Biochemical Pharmacology

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NADPH oxidase Nox4 is expressed in a wide range of tissues and plays a role in cellular signaling by providing reactive oxygen species (ROS) as intracellular messengers. Nox4 oxidase activity is thought to be constitutive and regulated at the transcriptional level; however, we challenge this point of view and suggest that specific quinone derivatives could modulate this activity. In fact, we demonstrated a significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ, tBuBQ, and duroquinone) observed in 3 different cellular models, HEK293E, T-REx™, and chondrocyte cell lines. Our results indicate that the effect is specific toward Nox4 versus Nox2. Furthermore, we showed that NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4 activity is also stimulated by reducing agents that possibly act by reducing the disulfide bridge (Cys226, Cys270) located in the extracellular E-loop of Nox4. Such model of Nox4 activity regulation could provide new insight into the understanding of the molecular mechanism of the electron transfer through the enzyme, i.e., its potential redox regulation, and could also define new therapeutic targets in diseases in which quinones and Nox4 are implicated

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Section: Discussionmentioning

confidence: 76%

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

Nguyen

Lardy

Rousset

et al. 2013

Biochemical Pharmacology

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“…Disulfide bonds in proteins have a free energy of Ϸ2-5 kcal/mol (26,27). The electrical free energy gained by movement of an S4 voltage sensor with three positive charges through an electrical potential of Ϫ200 mV is 13.8 kcal/mol (see SI Text for calculations of the energy and force of voltage sensor movement).…”

Section: Resultsmentioning

confidence: 99%

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

DeCaen

Yarov‐Yarovoy

Zhao

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

138

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The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. One structural model predicts sequential formation of ion pairs between the S4 gating charges and negative charges in neighboring S2 and S3 transmembrane segments during gating. Here, we show that paired cysteine substitutions for the third gating charge (R3) in S4 and D60 in S2 of the bacterial sodium channel NaChBac form a disulfide bond during activation, thus ''locking'' the S4 segment and inducing slow inactivation of the channel. Disulfide locking closely followed the kinetics and voltage dependence of activation and was reversed by hyperpolarization. Activation of D60C:R3C channels is favored compared with single cysteine mutants, and mutant cycle analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation.voltage-sensing ͉ gating charge ͉ mutant cycle ͉ sliding helix V oltage-gated ion channels conduct electrical currents that are essential for a wide range of physiological processes including neuronal excitation, action potential conduction, synaptic neurotransmission, and muscle contraction. In prokaryotes, ion channels are necessary for pH homeostasis, chemotaxis, and motility. Voltage-gated ion channels respond to changes in membrane potential by opening and closing (''gating'') their ion-conducting pathway across the cell membrane. Gating is rapid, reversible, and steeply voltage-dependent, suggesting that charged gating particles associated with the channels move across the membrane in response to the electric field (1). Capacitative ''gating currents'' caused by these charge movements (2, 3) indicate that approximately 3-4 positive charges per voltage-sensing module move outward during gating of sodium or potassium channels (4-10). The primary structure of sodium channels (11) revealed four homologous domains containing six predicted alpha-helical segments (S1-S6) in each. Subsequently, the positively charged S4 segment was proposed to have a transmembrane position, serve as the voltage sensor that perceives changes in membrane potential, and move outward along a spiral path during channel activation to conduct the gating current and initiate a conformational change to open the pore (12, 13). A major thermodynamic obstacle to transmembrane movement of the S4 segment is stabilization of its positively charged amino acid residues in the membrane environment. The sliding helix model posits (12) that sequential formation of ion pairs with negatively charged amino acid residues in the S1, S2, and/or S3 segments serve to stabilize the S4 segments in the membrane and thereby catalyze their transmembrane movement. A detailed structural version of th...

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“…Disulfide cross-linking strategies have proven to be a powerful tool to analyze the structures of the intermediates in protein folding reactions and of intermediate and fully activated states in enzymes (21)(22)(23). We previously demonstrated using a disulfide-locking method that the third gating charge (R3) in the S4 segment of NaChBac forms an ion pair with D60 near the extracellular end of the S2 segment (24).…”

mentioning

confidence: 99%

Sequential formation of ion pairs during activation of a sodium channel voltage sensor

DeCaen

Yarov‐Yarovoy

Sharp

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

135

143

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Electrical signaling in biology depends upon a unique electromechanical transduction process mediated by the S4 segments of voltage-gated ion channels. These transmembrane segments are driven outward by the force of the electric field on positively charged amino acid residues termed ''gating charges,'' which are positioned at three-residue intervals in the S4 transmembrane segment, and this movement is coupled to opening of the pore. Here, we use the disulfide-locking method to demonstrate sequential ion pair formation between the fourth gating charge in the S4 segment (R4) and two acidic residues in the S2 segment during activation. R4 interacts first with E70 at the intracellular end of the S2 segment and then with D60 near the extracellular end. Analysis with the Rosetta Membrane method reveals the 3-D structures of the gating pore as these ion pairs are formed sequentially to catalyze the S4 transmembrane movement required for voltagedependent activation. Our results directly demonstrate sequential ion pair formation that is an essential feature of the sliding helix model of voltage sensor function but is not compatible with the other widely discussed gating models.electrical excitability ͉ gating E lectrical signaling in biology depends upon a unique electromechanical transduction process that couples small changes in the electrical potential across the cell membrane to conformational changes that open and close the pores of voltage-gated ion channels. The high sensitivity of voltage-gated ion channels to small changes in membrane potential depends on gating charges within their transmembrane structure, which are driven outward across the membrane by changes in the electric field and trigger a series of conformational changes that result in opening the pore (1-4). Approximately 12-16 positive charges move across the membrane during activation of the voltage sensors of voltage-gated sodium or potassium channels (5-10). Two major thermodynamic obstacles that the voltage-sensing process must overcome are stabilization of amino acid residues that serve as gating charges in the transmembrane environment of the protein and catalysis of their outward transmembrane movement.Voltage-gated ion channels are composed of four subunits or domains that each contain six transmembrane segments (11). The S1-S4 segments serve as the voltage sensing module, and the S5 and S6 segment and their connecting P loop serve as the pore-forming module (2-4). The primary gating charges reside in the S4 segment in four to seven repeated motifs of a positively charged amino acid residue (usually arginine) followed by two hydrophobic residues (2-4). The crux of the problem of understanding the electromechanical coupling in voltage-gated ion channels is determining the structural and mechanistic basis for movement of these gating charges across the membrane and for coupling of this movement to pore opening. The sliding helix or helical screw models posit that sequential formation of ion pairs with negatively charged amino acid residues in t...

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Redox potentials of active-site bis(cysteinyl) fragments of thiol-protein oxidoreductases

Cited by 77 publications

References 40 publications

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

Sequential formation of ion pairs during activation of a sodium channel voltage sensor

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