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PubMed2005

Redox equilibria in hydroxylamine oxidoreductase. Electrostatic control of electron redistribution in multielectron oxidative processes.

We report results of continuum electrostatics calculations of the cofactor redox potentials, and of the titratable group pK(a) values, in hydroxylamine oxidoreductase (HAO). A picture of a sophisticated multicomponent control of electron flow in the protein emerged from the studies. First, we found that neighboring heme cofactors strongly interact electrostatically, with energies of 50-100 mV. Thus, cofactor redox potentials depend on the oxidation state of other cofactors, and cofactor redox potentials in the active (partially oxidized) enzyme differ substantially from the values obtained in electrochemical redox titration experiments. We found that, together, solvent-exposed heme 1 (having a large negative redox potential) and heme 2 (having a large positive redox potential) form a lock for electrons generated during the oxidation reaction The attachment of HAO's physiological electron transfer partner cytochrome c(554) results in a positive shift in the redox potential of heme 1, and "opens the electron gate". Electrons generated as a result of hydroxylamine oxidation travel to heme 3 and heme 8, which have redox potentials close to 0 mV versus NHE (this result is in partial disagreement with an existing experimental redox potential assignment). The closeness of hemes 3 and 8 from different enzyme subunits allows redistribution of the four electrons generated as a result of hydroxylamine oxidation, among the three enzyme subunits. For the multielectron oxidation process to be maximally efficient, the redox potentials of the electron-accepting cofactors should be roughly equal, and electrostatic interactions between extra electrons on these cofactors should be minimal. The redox potential assignments presented in the paper satisfy this general rule.

Bacterial Proteins↗
PubMed1999

Redox reactivity of animal apoferritins and apoheteropolymers assembled from recombinant heavy and light human chain ferritins.

The redox reactivities of air-oxidized apo horse spleen ferritin (HoSF) and apo rat liver ferritin (RaF) were examined by microcoulometry and reductive optical titrations. Microcoulometry on several independent lots of commercial HoSF revealed two distinct types of redox activity: one requiring 3-4 electrons and one requiring 6-7 electrons for full reduction of the protein shell. ApoRaF required 8-9 electrons to fully reduce the oxidized form. Reductive optical titrations confirmed the microcoulometric reduction stoichiometry and, in addition, showed that the spectra of both oxidized and reduced apoHoSF were distinct and possessed absorbances tailing into the visible region. The redox reactivity of both apoRaF and apoHoSF correlated with their H-subunit composition. Identical microcoulometric and optical experiments were conducted with recombinant apo human liver heavy (rHuHF) and light (rHuLF) ferritins, but neither was redox-active. These results suggest that the redox reactivity of native ferritins is due to their heteropolymeric nature. This was confirmed by mixing various proportions of rHuHF and rHuLF, dissociating the 24-mers into individual subunits with guanidine hydrochloride at pH 3.5, and renaturing to form heteropolymeric 24-mers. Microcoulometric measurements of these apoheteropolymers reassembled in vitro showed that they were redox-active like their native apoheteropolymer counterparts. The redox activity of these apoheteropolymers increased with H-subunit composition, reached a maximum near 12 H- and 12 L-subunits, and then declined to zero with increasing L-subunit composition. The decline in redox reactivity at high L-subunit concentrations indicates that both H- and L-subunits are involved in forming the observed redox centers. Apoheteropolymers formed from rHuLF and W93F (an H-chain mutant) were redox-inactive, suggesting that the conserved tryptophan is necessary for redox center formation.

Animals↗
PubMed2002

Coupled redox potentials in manganese and iron superoxide dismutases from reaction kinetics and density functional/electrostatics calculations.

A methodology for determining the coupled redox potentials (DeltaE(redox) degrees (coupled)) of manganese and iron superoxide dismutases (Mn(Fe)SODs), from the standard redox potential of reaction (O(2)(-) + 2H(+) + e(-) --> H(2)O(2)) and the experimental kinetic rate constants of Mn(Fe)SOD proteins, has been presented for the first time. A combined density functional (DF) and electrostatic protein/reaction field (DF/electrostatics) model has also been applied to seven protein structures, to study the structural, energetic, simple redox potential, pK(a), and coupled redox potential properties associated with each active site. The quantum cluster active site models, which include the metal, first shell ligands, represented by amino acid side chains and a solvent derived ligand, and the second shell H-bonding partners, were taken from the crystal structures, and geometry was optimized in four kinds of states: oxidized (III) and reduced (II) states with either a H(2)O molecule or a OH(-) group as the fifth coordinated ligand. We conclude from the calculations that the oxidized and reduced Mn(Fe)SODs are in the Mn(3+)(Fe(3+))(OH(-)) and Mn(2+)(Fe(2+))(H(2)O) forms, respectively; proton transfers will happen in both steps of the dismutation of superoxide anion (O(2)(-)), and the proton-transfer reactions will occur prior to or concerted with the electron transfer from O(2)(-) group to the Mn(3+)(Fe(3+))SOD metal center. The DeltaE(redox) degrees (coupled) of E. coli FeSOD calculated by the DF/electrostatics method is 0.16 V, which is very close to the experimental value of 0.25 V. The absolute values of DeltaE(redox) degrees (coupled) for T. thermophilus, human wild-type, and mutant Q143N MnSODs obtained from the DF/electrostatics method are -0.25, -0.29, and -0.11 V, which present the same trend and very similar relative values to those obtained from experimental kinetic rate constants (0.40, 0.32, and 0.59 V, respectively). The order DeltaE(redox) degrees (human wild-type) < DeltaE(redox) degrees (T. thermophilus) < DeltaE(redox) degrees (E. coli) < DeltaE(redox) degrees (Q143N) for MnSOD proteins is predicted by the DF/electrostatics calculations.

Binding Sites↗
PubMed2003

Evidence that SNF1-related kinase and hexokinase are involved in separate sugar-signalling pathways modulating post-translational redox activation of ADP-glucose pyrophosphorylase in potato tubers.

We recently discovered that post-translational redox modulation of ADP-glucose pyrophosphorylase (AGPase) is a powerful new mechanism to adjust the rate of starch synthesis to the availability of sucrose in growing potato tubers. A strong correlation was observed between the endogenous levels of sucrose and the redox-activation state of AGPase. To identify candidate components linking AGPase redox modulation to sugar supply, we used potato tuber discs as a model system. When the discs were cut from growing wild-type potato tubers and incubated for 2 h in the absence of sugars, redox activation of AGPase decreased because of a decrease in internal sugar levels. The decrease in AGPase redox activation could be prevented when glucose or sucrose was supplied to the discs. Both sucrose uptake and redox activation of AGPase were increased when EDTA was used to prepare the tuber discs. However, EDTA treatment of discs had no effect on glucose uptake. Feeding of different glucose analogues revealed that the phosphorylation of hexoses by hexokinase is an essential component in the glucose-dependent redox activation of AGPase. In contrast to this, feeding of the non-metabolisable sucrose analogue, palatinose, leads to a similar activation as with sucrose, indicating that metabolism of sucrose is not necessary in the sucrose-dependent AGPase activation. The influence of sucrose and glucose on redox activation of AGPase was also investigated in discs cut from tubers of antisense plants with reduced SNF1-related protein kinase activity (SnRK1). Feeding of sucrose to tuber discs prevented AGPase redox inactivation in the wild type but not in SnRK1 antisense lines. However, feeding of glucose leads to a similar activation of AGPase in the wild type and in SnRK1 transformants. AGPase redox activation was also increased in transgenic tubers with ectopic overexpression of invertase, containing high levels of glucose and low sucrose levels. Expression of a bacterial glucokinase in the invertase-expressing background led to a decrease in AGPase activation state and tuber starch content. These results show that both sucrose and glucose lead to post-translational redox activation of AGPase, and that they do this by two different pathways involving SnRK1 and an endogenous hexokinase, respectively.

Enzyme Activation↗
PubMedDate not supplied

Zinc coordination environments in proteins as redox sensors and signal transducers.

Zinc/cysteine coordination environments in proteins are redox-active. Oxidation of the sulfur ligands mobilizes zinc, while reduction of the oxidized ligands enhances zinc binding, providing redox control over the availability of zinc ions. Some zinc proteins are redox sensors, in which zinc release is coupled to conformational changes that control varied functions such as enzymatic activity, binding interactions, and molecular chaperone activity. Whereas the released zinc ion in redox sensors has no known function, the redox signal is transduced to specific and sensitive zinc signals in redox transducers. Released zinc can bind to sites on other proteins and modulate signal transduction, generation of metabolic energy, mitochondrial function, and gene expression. The paradigm of such redox transducers is the zinc protein metallothionein, which, together with its apoprotein, thionein, functions at a central node in cellular signaling by redistributing cellular zinc, presiding over the availability of zinc, and interconverting redox and zinc signals. In this regard, the transduction of nitric oxide (NO) signals into zinc signals by metallothionein has received particular attention. It appears that redox-inert zinc has been chosen to control some aspects of cellular thiol/disulfide redox metabolism. Tight control of zinc is essential for redox homeostasis because both increases and decreases of cellular zinc elicit oxidative stress. Depending on its availability, zinc can be cytoprotective as a pro-antioxidant or cytotoxic as a pro-oxidant. Any condition with acute or chronic oxidative stress is expected to perturb zinc homeostasis.

Animals↗
PubMed2006

Extracellular redox state: refining the definition of oxidative stress in aging.

Oxidative stress in aging can result from an imbalance of prooxidants and antioxidants with excessive, destructive free radical chemistry. Thiol systems are important in the control of these processes, both by protecting against damage and serving in redox signaling mechanisms to sense danger and repair the damage. Studies by a number of research groups in collaboration with the Emory Clinical Biomarkers Laboratory show that the redox state of the central tissue antioxidant, glutathione (GSH), can be measured in human plasma and provides a quantitative systemic indicator of oxidative stress. Plasma GSH/GSSG redox in humans becomes oxidized with age, in response to chemotherapy, as a consequence of cigarette smoking, and in association with common age-related diseases (e.g., type 2 diabetes, cardiovascular disease). However, the GSH/GSSG redox is not equilibrated with the larger plasma cysteine/cystine (Cys/CySS) pool, and the Cys/CySS redox varies with age in a pattern that is distinct from that of GSH/GSSG redox. Furthermore, in vitro studies show that variation in Cys/CySS redox over the range found in vivo affects signaling pathways, which control cell proliferation and oxidant-induced apoptosis. The results point to the conclusion that free radical scavenging antioxidants are of increased importance when thiol/disulfide redox states are oxidized. Because thiol/disulfide redox states, per se, function in redox signaling and control as well as antioxidant protection, GSH/GSSG and Cys/CySS redox states may provide central parameters to link environmental influences and progression of changes associated with aging.

Aging↗
PubMed2005

Extracellular cysteine/cystine redox regulates the p44/p42 MAPK pathway by metalloproteinase-dependent epidermal growth factor receptor signaling.

Previous research shows that stimulation of proliferation of colon carcinoma (Caco-2) cells by a more reduced extracellular cysteine/cystine (Cys/CySS) redox state occurs with no apparent effect on intracellular glutathione and that this stimulation is lost on addition of epidermal growth factor. The purpose of the present study was to determine whether a more reduced extracellular Cys/CySS redox state activates the mitogenic p44/p42 mitogen-activated protein kinase (MAPK) pathway and whether this is signaled through the epidermal growth factor receptor (EGFR). Caco-2 cells were exposed to a range of physiological extracellular redox conditions from -150 to 0 mV. In the absence of added growth factors, the most reduced (-150 mV) redox state induced an 80% increase in EGFR phosphorylation, and this was followed by a marked increase in phosphorylation of p44/p42 MAPK. Inhibitors of EGFR (AG1478) and p44/p42 MAPK (U0126) phosphorylation blocked redox-dependent p44/p42 phosphorylation, indicating that signaling occurred by EGFR. These effects were inhibited by pretreatment with a nonpermeant alkylating agent, showing that signaling involved thiols accessible to the extracellular space. The EGFR ligand TGF-alpha was increased in culture medium at more reduced redox states. Redox-dependent phosphorylation of EGFR was completely prevented by a metalloproteinase inhibitor (GM6001), and an antibody to TGF-alpha partially inhibited the phosphorylation of p44/p42 MAPK by redox. Thus the data show that a redox-dependent activation of metalloproteinase can stimulate the mitogenic p44/p42 MAPK pathway by a TGF-alpha-dependent mechanism. Because Cys availability and Cys/CySS redox are dependent on nutrition, disease, and environmental exposures, the results suggest that cell proliferation could be influenced physiologically by Cys-dependent redox effects on growth factor signaling pathways.

Antimetabolites↗
PubMed1997

Redox state changes in density-dependent regulation of proliferation.

The ability of certain transcription factors to bind to DNA has been demonstrated to be influenced by the redox environment. Therefore, fluctuations in the redox state of the cell may regulate the transcription of genes which control proliferation. To assess whether changes in the redox state may be related to proliferation, levels of oxidized (GSSG) and reduced (GSH) glutathione, the primary modulators of the redox state, were measured in cultures of varying densities of normal human fibroblasts which exhibit contact inhibition of proliferation, as well as fibrosarcoma cells, which lack this mechanism of growth control. Redox potentials calculated from normal, proliferating fibroblasts were found to be -34 mV more reducing than confluent, contact-inhibited cells. However, fibrosarcoma cells did not demonstrate this modulation in redox state. Further, to delineate whether these redox changes were the consequence or the cause of contact inhibition, cultures of subconfluent proliferating fibroblasts were treated with modulators of glutathione synthesis. Buthionine sulfoximine, an inhibitor of GSH synthesis, induced a less reducing redox state and decreased proliferation. In contrast, GSH synthesis precursors caused a more reduced redox state and increased proliferation. Collectively, these results suggest an interrelationship between redox state and growth control.

Acetylcysteine↗
PubMed2006

Redox regulation of cardiac calcium channels and transporters.

Intracellular concentrations of redox-active molecules can significantly increase in the heart as a result of activation of specific signal transduction pathways or the development of certain pathophysiological conditions. Changes in the intracellular redox environment can affect many cellular processes, including the gating properties of ion channels and the activity of ion transporters. Because cardiac contraction is highly dependent on intracellular Ca(2+) levels ([Ca(2+)](i)) and [Ca(2+)](i) regulation, redox modification of Ca(2+) channels and transporters has a profound effect on cardiac function. The sarcoplasmic reticulum (SR) Ca(2+) release channel, or ryanodine receptor (RyR), is one of the well-characterized redox-sensitive ion channels in the heart. The redox modulation of RyR activity is mediated by the redox modification of sulfhydryl groups of cysteine residues. Other key components of cardiac excitation-contraction (e-c) coupling such as the SR Ca(2+) ATPase and L-type Ca(2+) channel are subject to redox modulation. Redox-mediated alteration of the activity of ion channels and pumps is directly involved in cardiac pathologies such as ischemia-reperfusion injury. Significant bursts of reactive oxygen species (ROS) generation occur during reperfusion of the ischemic heart, and changes in the activity of the major components of [Ca(2+)](i) regulation, such as RyR, Na(+)-Ca(2+) exchange and Ca(2+) ATPases, are likely to play an important role in ischemia-related Ca(2+) overload. This article summarizes recent findings on redox regulation of cardiac Ca(2+) transport systems and discusses contributions of this redox regulation to normal and pathological cardiac function.

Animals↗
PubMed1996

Structural origins of redox potentials in Fe-S proteins: electrostatic potentials of crystal structures.

Redox potentials often differ dramatically for homologous proteins that have identical redox centers. For two types of iron-sulfur proteins, the rubredoxins and the high-potential iron-sulfur proteins (HiPIPs), no structural explanations for these differences have been found. We calculated the classical electrostatic potential at the redox site using static crystal structures of four rubredoxins and four HiPIPs to identify important structural determinants of their redox potentials. The contributions from just the backbone and polar side chains are shown to explain major features of the experimental redox potentials. For instance, in the rubredoxins, the presence of Val 44 versus Ala 44 causes a backbone shift that explains a approximately 50 mV lower redox potential in one of the four rubredoxins. This result is consistent with experimental redox potentials of five additional rubredoxins with known sequence. Also, we attribute the unusually lower redox potentials of two of the HiPIPs studied to a less positive electrostatic environment around their redox sites. Finally, molecular dynamics simulations of solvent around static rubredoxin crystal structures indicate that water alone is a major factor in dampening the contribution of charged side chains, in accord with experiments showing that mutations of surface charges produce relatively little effect on redox potentials.

Alanine↗
PubMed2000

Cellular thiols and redox-regulated signal transduction.

In contrast to the conventional notion that reactive oxygen is mostly a trigger for oxidative damage of biological structures, now we know that low physiologically relevant concentrations of ROS can regulate a variety of key molecular mechanisms that may be linked with important cell functions (Fig. 4). Redox-based regulation of gene expression has emerged as a fundamental regulatory mechanism in cell biology. Several proteins, with apparent redox-sensing activity, have been described. Electron flow through side-chain functional CH2-SH groups of conserved cysteinyl residues in these proteins account for the redox-sensing properties. Protein thiol groups with high thiol-disulfide oxidation potentials are likely to be redox-sensitive. The ubiquitous endogenous thiols thioredoxin and glutathione are of central importance in redox signaling. Signals are transduced from the cell surface to the nucleus through phosphorylation and dephosphorylation chain reactions of cellular proteins at tyrosine and serine/threonine. Protein phosphorylation, one of the most fundamental mediators of cell signaling, is redox-sensitive. DNA-binding proteins are involved in the regulation of cellular processes such as replication, recombination, viral integration and transcription. Several studies show that the interaction of certain transcription regulatory proteins with their respective cognate DNA sites is also redox-regulated. Changes in the concentration of Ca2+i control a wide variety of cellular functions, including transcription and gene expression; Ca(2+)-driven protein phosphorylation and proteolytic processing of proteins are two major intracellular events that are implicated in signal transduction from the cell surface to the nucleus. Intracellular calcium homeostasis is regulated by the redox state of cellular thiols, and it is evident that cell calcium may play a critical role in the activation of the redox-sensitive transcription factor NF-kappa B. Among the several thiol agents tested for their efficacy in modulating cellular redox status, N-acetyl-L-cysteine and alpha-lipoic acid hold most promise for human use. A strong therapeutic potential of strategies that would modulate the cellular thioredoxin system has been also evident.

Animals↗
PubMed2006

Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy.

Electrochemical redox titrations of cytochrome c oxidase from Paraccocus denitrificans were performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The majority of the differential infrared absorption features may be divided into four groups, which correlate with the redox transitions of the four redox centers of the enzyme. Infrared spectroscopy has the advantage of allowing one to measure independent alterations in redox centers, which are not well separated, or even observed, by other spectroscopic techniques. We found 12 infrared bands that titrated with the highest observed midpoint redox potential (E(m) = 412 mV at pH 6.5) and which had a pH dependence of 52 mV per pH unit in the alkaline region. These bands were assigned to be linked to the Cu(B) center. We assigned bands to the Cu(A) center that showed a pH-independent E(m) of 250 mV. Two other groups of infrared differential bands reflected redox transitions of the two heme groups and showed a more complex behavior. Each of them included two parts, corresponding to high- and low-potential redox transitions. For the bands representing heme a, the ratio of high- to low-potential components was ca. 3:2; for heme a(3) this ratio was ca. 2:3. Taking into account the redox interactions between the hemes, these ratios yielded a difference in E(m) of 9 mV between the hemes (359 mV for heme a; 350 mV for heme a(3) at pH 8.0). The extent of the redox interaction between the hemes (-115 mV at pH 8.0) was found to be pH-dependent. The pH dependence of the E(m) values for the two hemes was the same and about two times smaller than the theoretical one, suggesting that an acid/base group binds a proton upon reduction of either heme. The applied approach allowed assignment of infrared bands in each of the four groups to vibrations of the hemes, ligands of the redox centers, amino acid residues, and/or protein backbone. For example, the well-known band shift at 1737/1746 cm(-)(1) corresponding to the protonated glutamic acid E278 correlated with oxidoreduction of heme a.

Copper↗
PubMed1996

Behavioral responses of Escherichia coli to changes in redox potential.

Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential. In a spatial redox gradient of benzoquinone/benzoquinol, E. coli cells migrated to form a sharply defined band. Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band. This behavioral response was named redox taxis. Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force. The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector. The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis. A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis. A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein. A similar mechanism has been proposed for aerotaxis. Redox taxis may play an important role in the distribution of bacterial species in natural environments.

Bacterial Proteins↗
PubMed2002

Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging.

PURPOSE: To test the feasibility of electron paramagnetic resonance imaging (EPRI) to provide non-invasive images of tissue redox status using redox-sensitive paramagnetic contrast agents. MATERIAL AND METHODS: Nitroxide free radicals were used as paramagnetic agents and a custom-built 300 MHz EPR spectrometer/imager was used for all studies. A phantom was constructed consisting of four tubes containing equal concentrations of a nitroxide. Varying concentrations of hypoxanthine/xanthine oxidase were added to each tube and reduction of the nitroxide was monitored by EPR as a function of time. Tumor-bearing mice were intravenously infused with a nitroxide and the corresponding reduction rate was monitored on a pixel-by-pixel basis using 2D EPR of the tumor-bearing leg and normal leg serving as control. For animal studies, nitroxides were injected intravenously (1.25 mmol/kg) and EPR projections were collected every 3 min after injection using a magnetic field gradient of 2.5 G/cm. The reduction rates of signal intensity on a pixel-by-pixel basis were calculated and plotted as a redox map. Redox maps were also collected from the mice treated with diethylmaleate (DEM), which depletes tissue thiols and alters the global redox status. RESULTS: Redox maps obtained from the phantoms were in agreement with the intensity change in each of the tubes where the signals were decreasing as a function of the enzymatic activity, validating the ability of EPRI to accurately access changes in nitroxide reduction. Redox imaging capability of EPR was next evaluated in vivo. EPR images of the nitroxide distribution and reduction rates in tumor-bearing leg of mice exhibited more heterogeneity than in the normal tissue. Reduction rates were found to be significantly decreased in tumors of mice treated with DEM, consistent with the depletion of thiols and the consequent alteration of the redox status. CONCLUSION: Using redox-sensitive paramagnetic contrast agents, EPRI can non-invasively discriminate redox status differences between normal tissue and tumors.

Animals↗
PubMed2002

CxxS: fold-independent redox motif revealed by genome-wide searches for thiol/disulfide oxidoreductase function.

Redox reactions involving thiol groups in proteins are major participants in cellular redox regulation and antioxidant defense. Although mechanistically similar, thiol-dependent redox processes are catalyzed by structurally distinct families of enzymes, which are difficult to identify by available protein function prediction programs. Herein, we identified a functional motif, CxxS (cysteine separated from serine by two other residues), that was often conserved in redox enzymes, but rarely in other proteins. Analyses of complete Escherichia coli, Campylobacter jejuni, Methanococcus jannaschii, and Saccharomyces cerevisiae genomes revealed a high proportion of proteins known to use the CxxS motif for redox function. This allowed us to make predictions in regard to redox function and identity of redox groups for several proteins whose function previously was not known. Many proteins containing the CxxS motif had a thioredoxin fold, but other structural folds were also present, and CxxS was often located in these proteins upstream of an alpha-helix. Thus, a conserved CxxS sequence followed by an alpha-helix is typically indicative of a redox function and corresponds to thiol-dependent redox sites in proteins. The data also indicate a general approach of genome-wide identification of redox proteins by searching for simple conserved motifs within secondary structure patterns.

Amino Acid Motifs↗
PubMed1981

An analysis of activity determinations in a series of coupled redox reactions with special reference to hydrogenase.

In this paper a spectrophotometric method is described to study the effects of pH and redox potential on the hydrogen production activity of hydrogenase, which will be applied in the following paper. The theoretical part of this paper gives the mathematical description of a system in which a redox mediator is continuously reduced by a redox system, in a non-rate-limiting way and continuously enzymatically oxidized, in a rate-limiting way. Due to the changes in redox potential during the course of the reaction the ratio reduced/oxidized redox mediator declines gradually. It is possible to relate the changes in this ratio to the actual amount of product formed. The calculation of product formation has been applied to the production of hydrogen by hydrogenase in a system with a redox mediator, whose reduced form functions as the ultimate electron donor for hydrogenase, and the dithionite/(bi)sulphite redox couple as reducing system. The changes in the ratio reduced/oxidized redox mediator as a function of pH and redox potential, followed spectrophotometrically, have been related to the hydrogen production activity of hydrogenase. The second part of the paper deals with the analysis of the theory and describes the conditions under which the method can be used with methyl viologen as redox mediator. The conditions for determining the pH dependence of the hydrogen production activity manometrically are also described.

Dithionite↗
PubMed1995

Carotene desaturation is linked to a respiratory redox pathway in Narcissus pseudonarcissus chromoplast membranes. Involvement of a 23-kDa oxygen-evolving-complex-like protein.

The enzymic activity of phytoene desaturase in Narcissus pseudonarcissus chromoplast membranes depends in an essential way on the redox state of its environment. Here, the main redox-active components are quinones and tocopherols. Quinones (oxidized) act as intermediate electron acceptors in the desaturation reaction, as can be shown in reduced, hydroquinone-rich membranes. However, their complete oxidation by ferricyanide treatment of membranes leads to inhibition of the desaturation activity and, under these conditions, hydroquinones are required for reactivation. Using redox titrations, it is shown here that the optimal activity lies in the range of the midpoint potential of the plastoquinone/plastohydroquinone redox couple. For the adjustment of redox states of the redox-active lipid components in (photosynthetically inactive) chromoplasts, NADPH and oxygen are involved, the latter acting as a terminal acceptor. This results in a respiratory redox pathway in chromoplast membranes which is described here, to our knowledge, for the first time. Since phytoene desaturation responds to the redox state of quinones, which is adjusted by the respiratory redox pathway, the two reactions must be regarded as being mechanistically linked. The first protein component involved in the respiratory pathway which we have investigated molecularly is a 43-kDa NAD(P)H:quinone oxidoreductase, which is organized as a homodimer (23 +/- 3 kDa/subunit) and apparently possesses a manganese redox center. Internal protein microsequencing and cloning of the corresponding cDNA revealed a high degree of similarity to the 23-kDa protein of the oxygen-evolving complex of photosystem II, but no information about the N-terminal organization of the oxidoreductase could be obtained. During flower development, the steady-state concentration of the corresponding mRNA is up-regulated, indicating a specific function of the gene product in chlorophyll-free chromoplasts.

Amino Acid Sequence↗
PubMed2005

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells.

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP(+)/reduced NADP and NAD(+)/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD(+)/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples.

Adenosine Diphosphate↗