Topography of human cytochrome b5/cytochrome b5 reductase interacting domain and redox alterations upon complex formation

Cytochrome b₅ is the main electron acceptor of cytochrome b₅ reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b₅ reductase flavin autofluorescence that was dependent upon the presence of cytochrome b_5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5 ± 0.1 μM and a 1:1 stoichiometry for the complex formation. In addition, a 30 mV negative shift of cytochrome b₅ reductase redox potential in presence of cytochrome b₅ was also measured. These experiments suggest that the FAD group of cytochrome b₅ reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins.     

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b,iron-sulfur protein and cytochrome c1

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc₁. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Q_o site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c₁, iii) reduction of cytochrome c₁ by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c₁.In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c₁ and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c₁ of cytochrome c₁, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc₁ were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Q_o site and cytochrome c₁ helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Q_o site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.     

RegB Kinase Activity Is Repressed by Oxidative Formation of Cysteine Sulfenic Acid

RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.     

Green tea catechins can bind and modify ERp57/PDIA3 activity

Background

Green tea is a rich source of polyphenols, mainly catechins (flavanols), which significantly contribute to the beneficial health effects of green tea in the prevention and treatment of various diseases. In this study the effects of four green tea catechins on protein ERp57, also known as protein disulfide isomerase isoform A3 (PDIA3), have been investigated in an in vitro model.

Methods

The interaction of catechins with ERp57 was explored by fluorescence quenching and surface plasmon resonance techniques and their effect on ERp57 activities was investigated.

Results

A higher affinity was observed for galloylated cathechins, which bind close to the thioredoxin-like redox-sensitive active sites of the protein, with a preference for the oxidized form. The effects of these catechins on ERp57 properties were also investigated and a moderate inhibition of the reductase activity of ERp57 was observed as well as a strong inhibition of ERp57 DNA binding activity.

Conclusions

Considering the high affinity of galloylated catechins for ERp57 and their capability to inhibit ERp57 binding to other macromolecular ligands, some effects of catechins interaction with this protein on eukaryotic cells may be expected.

General significance

This study provides information to better understand the molecular mechanisms underlying the biological activities of catechins and to design new polyphenol-based ERp57-specific inhibitors.     

Identification of potential protein dithiol-disulfide substrates of mammalian Grx2

Background

Glutaredoxins (Grxs) catalyze the reduction of protein disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism. These rapid, specific, and generally also reversible modifications are part of various signaling cascades regulating for instance cell proliferation, differentiation and apoptosis. Even though crucial functions of the conserved, mitochondrial Grx2a and the cytosolic/nuclear Grx2c isoforms have been proposed, only a few substrates have been identified in vitro or in vivo. The significance of redox signaling is emerging, yet a general lack of methods for the time-resolved analysis of these distinct and rapid modifications in vivo constitutes the biggest challenge in the redox signaling field.

Methods and results

Here, we have identified potential interaction partners for Grx2 isoforms in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates are part of the cytoskeleton or act in protein folding, cellular signaling and metabolism. Part of these interactions were further verified by immunoprecipitation or a newly established 2-D redox blot.

Conclusions

Our study demonstrates that Grx2 catalyzes both the specific oxidation and the reduction of cysteinyl residues in the same compartment at the same time and without affecting the global cellular thiol-redox state.

General significance

The knowledge of specific targets will be helpful in understanding the functions of Grx2. The 2-D redox blot may be useful for the analysis of the overall thiol-redox state of proteins with high molecular weight and numerous cysteinyl residues, that evaded analysis by previously described methods.     

Generating S-Nitrosothiols from Hemoglobin: MECHANISMS,CONFORMATIONAL DEPENDENCE,AND PHYSIOLOGICAL RELEVANCE*

In vitro, ferrous deoxy-hemes in hemoglobin (Hb) react with nitrite to generate nitric oxide (NO) through a nitrite reductase reaction. In vivo studies indicate Hb with nitrite can be a source of NO bioactivity. The nitrite reductase reaction does not appear to account fully for this activity because free NO is short lived especially within the red blood cell. Thus, the exporting of NO bioactivity both out of the RBC and over a large distance requires an additional mechanism. A nitrite anhydrase (NA) reaction in which N₂O₃, a potent S-nitrosating agent, is produced through the reaction of NO with ferric heme-bound nitrite has been proposed (Basu, S., Grubina, R., Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang, A., He, X., Azarov, I., Seibert, R., Mehta, A., Patel, R., King, S. B., Hogg, N., Ghosh, A., Gladwin, M. T., and Kim-Shapiro, D. B. (2007) Nat. Chem. Biol. 3, 785–794) as a possible mechanism. Legitimate concerns, including physiological relevance and the nature of the mechanism, have been raised concerning the NA reaction. This study addresses these concerns demonstrating NO and nitrite with ferric hemes under near physiological conditions yield an intermediate having the properties of the purported NA heme-bound N₂O₃ intermediate. The results indicate that ferric heme sites, traditionally viewed as a source of potential toxicity, can be functionally significant, especially for partially oxygenated/partially met-R state Hb that arises from the NO dioxygenation reaction. In the presence of low levels of nitrite and either NO or a suitable reductant such as l-cysteine, these ferric heme sites can function as a generator for the formation of S-nitrosothiols such as S-nitrosoglutathione and, as such, should be considered as a source of RBC-derived and exportable bioactive NO.     

Thioredoxin and Thioredoxin Reductase Control Tissue Factor Activity by Thiol Redox-dependent Mechanism

Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples. The association is dependent on hTrx1-Cys-73 that bridges TF-Cys-209 via a disulfide bond. hTrx1-Cys-73 is absolutely required for hTrx1 to interfere with FVIIa binding to purified and cell-surface TF, consequently suppressing TF-dependent procoagulant activity and proteinase-activated receptor-2 activation. Moreover, hTrx1/TrxR plays an important role in sensing the alterations of NADPH/NADP⁺ states and transducing this redox-sensitive signal into changes in TF activity. With NADPH, hTrx1/TrxR readily facilitates the reduction of TF, causing a decrease in TF activity, whereas with NADP⁺, hTrx1/TrxR promotes the oxidation of TF, leading to an increase in TF activity. By comparison, TF is more likely to favor the reduction by hTrx1-TrxR-NADPH. This reversible reduction-oxidation reaction occurs in the TF extracellular domain that contains partially opened Cys-49/-57 and Cys-186/-209 disulfide bonds. The cell-surface TF procoagulant activity is significantly increased after hTrx1-knockdown. The response of cell-surface TF procoagulant activity to H₂O₂ is efficiently suppressed through elevating cellular TrxR activity via selenium supplementation. Our data provide a novel mechanism for redox regulation of TF activity. By modifying Cys residues or regulating Cys redox states in TF extracellular domain, hTrx1/TrxR function as a safeguard against inappropriate TF activity.     

Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel

Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H⁺ (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys → Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome.