Redox Equivalents Research Articles

Electrochemical Regeneration of Oxidoreductases for Cell-free Biocatalytic Redox Reactions

Oxidoreductases represent a highly interesting and versatile class of biocatalysts for specific reduction, oxidation, and oxyfunctionalization reactions. Since oxidoreductases depend on cofactors and coenzymes to supply or withdraw redox equivalents released during the catalytic process, their application in cell-free environments requires external supply with these redox equivalents. Next to enzymatic approaches, a variety of non-enzymatic regeneration strategies have been developed. This review focuses on electrochemical methods for the in situ regeneration of nicotinamide cofactors as well as flavin- and heme-coenzymes, developed for synthetic application. The fields of electrochemical biosensors as well as biofuel cells are not discussed in detail. Electrochemical approaches bear much promise and in some cases are more efficient and more versatile than enzymatic regeneration approaches.

Metabolic regulation of potassium channels.

Potassium (K+) channels exist in all three domains of organisms: eubacteria, archaebacteria, and eukaryotes. In higher animals, these membrane proteins participate in a multitude of critical physiological processes, including food and fluid intake, locomotion, stress response, and cognitive functions. Metabolic regulatory factors such as O2, CO2/pH, redox equivalents, glucose/ATP/ADP, hormones, eicosanoids, cell volume, and electrolytes regulate a diverse group of K+ channels to maintain homeostasis.

Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism.

Primary leaf metabolism requires the co-ordinated production and use of carbon skeletons and redox equivalents in several subcellular compartments. The role of the mitochondria in leaf metabolism has long been recognized, but it is only recently that molecular tools and mutants have become available to evaluate cause-and-effect relationships. In particular, analysis of the CMSII mutant of Nicotiana sylvestris, which lacks functional complex I, has provided information on the role of mitochondrial electron transport in leaf function. The essential feature of CMSII is the absence of a major NADH sink, i.e. complex I. This necessitates re-adjustment of whole-cell redox homeostasis, gene expression, and also influences metabolic pathways that use pyridine nucleotides. In air, CMSII is not able to use its photosynthetic capacity as well as the wild type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen species, and enhanced stress resistance. Lastly, the loss of a major mitochondrial dehydrogenase has important repercussions for the integration of primary carbon and nitrogen metabolism, causing distinct changes in leaf organic acid profiles, and also affecting downstream processes such as the biosynthesis of the spectrum of leaf amino acids.

Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection.

Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50 of 32 microM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein.

Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1). Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

Differential response of genes for ferredoxin and ferredoxin : NADP + oxidoreductase to nitrate and light in maize leaves

In higher plants, ferredoxin (Fd) and Fd:NADP+ oxidoreductase (FNR, EC 1.18.1.2) are encoded by small multigene families, and the individuals transfer electrons to the dependent enzymes in the photosynthetic and the non-photosynthetic plastids. In maize, a C4 plant, expression of genes for the non-photosynthetic isoproteins, Fd VI and R-FNR, is responsive to nitrate in roots whereas the expression and the spatial distribution in the leaves have not been analysed. Here, we studied the expression pattern of a series of Fd and FNR genes in maize leaves in response to nitrate and light. Upon addition of nitrate, the transcripts for Fd VI and R-FNR rapidly accumulated in the leaves, whereas light did not induce accumulation. Expression of genes for the other isoproteins was not changed significantly by the nitrogen source. In the leaf, the transcripts for Fd VI and R-FNR were predominantly detected in mesophyll cells as were those for nitrate-assimilatory enzymes. Since R-FNR is an isoprotein transferring electrons from NADPH to non-photosynthetic type Fd, the redox equivalent is supplied in nitrate reduction, at least partially, via an oxidative pentose phosphate pathway, even in photosynthetic organs.

Pd-mediated synthesis of linked conjugated tri- and penta-ferrocenyl complexes

A facile synthesis of new conjugated oligo-ferrocenyl compounds is described. The synthetic method consists of a two step procedure, which combines olefination by the Wittig procedure and Pd-mediated CC coupling, leading to high yields of tri- and penta-ferrocenyl complexes. The crystal structures of the 1,1′-bissubstituted ferrocenyl precursors are described. The electrochemical analysis of the compounds obtained, reveals that the peripheral ferrocenyl units display an equivalent redox behavior, with a large separation of the peak corresponding to the central ferrocenyl unit.

VOCs – helping trees withstand global change?

VOCs – helping trees withstand global change?

PM and PR forms of cytochrome c oxidase have different spectral properties

The reaction between bovine heart cytochrome c oxidase and dioxygen was monitored at room temperature in the visible and Soret regions following photolysis of the mixed-valence CO-bound enzyme. Time-resolved optical absorption difference spectra were collected between 50 ns and 1.7 ms by a gated multichannel analyzer. Singular value decomposition and global exponential fitting resolved three processes with apparent lifetimes of 2.2±0.5, 17±4 and 160±30 μs. The spectra of the intermediates were extracted based on a sequential kinetic mechanism and compared to the corresponding intermediate spectra observed during the reaction of the fully reduced enzyme with dioxygen. The first process is associated with a conformational change at heme a3 upon dissociation of CO from CuB+ and concomitant back-electron transfer from heme a3 to heme a. This is followed by O2 binding to heme a3 forming compound A (AM), with a spectrum identical to that observed upon O2 binding to heme a3 in the fully reduced enzyme (AR). Intermediate AM decays into PM, the spectrum of which is equivalent to that of the 607 nm form, generated upon addition of H2O2 to the oxidized enzyme at alkaline pH values (PH). However, the spectrum of PM is significantly different from the corresponding intermediate observed upon the reaction of dioxygen with the fully reduced enzyme (PR). The spectral differences between PM and PR may arise from the different number of redox equivalents at the binuclear site, with a tyrosine radical in the PM state, and tyrosine or tyrosinate in PR, or may be the consequence of a more complex reaction mechanism in the case of the fully reduced enzyme.

Stoichiometric redox titrations of complex metalloenzymes

This chapter describes stoichiometric redox titration method and points out difficulties and avoidable pitfalls. This method may provide reliable evidence for a spectroscopically silent redox center in an enzyme, as long as the reduction potential of the center is within the potential range probed. Such centers may consume redox equivalents and thus alter the shape of the titration curves of spectroscopically active centers. Spectroscopically observable stable redox states are commonly viewed as being more important than those which cannot be easily observed, but this need not be the case. Thus, the results of stoichiometric titration studies may be critical for formulating the correct mechanism of catalysis. Finally, despite best efforts, some uncertainty in the results and analysis will inevitably remain. This ultimately translates into some persistent uncertainty regarding the redox/spectroscopic system present in the enzyme.

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica.

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH.

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.

Effect of the hemoregulatory peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1alpha is reduced in bone marrow cultures from patients with chronic myeloid leukemia (CML).

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.

Extensive redox series in dinuclear and dendritic Ru(II) complexes

The electrochemical behavior of a dinuclear and a hexanuclear dendritic ruthenium(II) bipyridine complexes with 2,3-bis(2-pyridyl)pyrazine (2,3-dpp) as bridging ligands has been investigated in highly purified liquid SO 2 or N, N-dimethylformamide (DMF) solutions. The compounds have general formula [Ru n (bpy) n+2 (2,3-dpp) n−1 ] 2 n+ , where n=2 or 6; bpy is 2,2′-bipyridine. The wide anodic and cathodic potential windows explored in liquid SO 2 and DMF, at low temperature, (up to ca. 4.3 and −3.1 V vs. SCE, respectively) have allowed the observation of several metal-centered oxidations and ligand-centered oxidations and reductions, leading to the most extensive redox series so far reported, comprising up to 26 reversible ligand-centered reductions and 14 reversible metal- or ligand-centered oxidations, for a total of 40 redox processes in the hexanuclear complex. The redox standard potentials for overlapping processes in multielectron waves have been obtained from the analysis of the voltammetric curves and their digital simulation, and the localization of all the redox processes and an evaluation of the mutual interactions between the redox centers have been obtained. These results allowed to clarify the important role played by bridging ligands in mediating the interactions between the equivalent redox sites. Furthermore, the localization of the redox sites and the determination of relative standard potentials allowed the mapping of MLCT excited states within the dendrimeric structure, through the correlation between the absorption spectroscopic data and the relevant voltammetric data.

Synthesis, Structure, and Electrochemistry of Mononuclear and Face-to-Face Binuclear Orthometalated Complexes of Palladium(II) with N-Monodentate or N(1),N(3)-Bridging 1,3-Di-p-tolyltriazenido Ligands. Dependence on Geometrical Arrangement of the Electronic Communication between Two Equivalent Redox Sites

The synthesis, electrochemistry, and structural characterization of the mononuclear complex [Pd{C6H4N(H)NC(CH3)C5H4N}(p-tolN−NNp-tol)] (1) containing the monodentate 1,3-di-p-tolyltriazenido ligand is described. Compound 1 is an example of a stable species containing a Pd−N amido bond cis to a Pd−C aryl bond. Kinetic parameters for the dynamic intramolecular N(1)−N(3) exchange of the monodentate ligand in complex 1 have been calculated. The cis and trans isomers of the orthometalated face-to-face complex [{Pd(C6H4NNC6H5)(μ-p-tolNNNp-tol)}2] (2) have also been prepared, and the crystal structure of the trans isomer is reported. There are noticeable differences in the electrochemical behavior of the mononuclear and binuclear species. From the electrochemical experiments on both isomers of 2 it is possible to recognize different redox sites, to calculate the electronic coupling between them, and to suggest where the reversible electron transfers occur. Each isomers of 2 undergoes two one-electron oxidations and two one-electron reductions. The electronic coupling (ΔE = 0.40 V) at oxidizing potentials is identical for both isomers of complex 2, suggesting that the oxidations occur on the Pd(μ-triazenido)2Pd framework which is common to both isomers. By contrast, the electronic coupling at reducing potentials is greater for cis-2 (ΔE = 0.33 V) than for trans-2 (ΔE = 0.25 V), suggesting that the reduction processes occur on the orthopalladated fragments, which are arranged differently on the two isomers. Thus, electronic communication between two equivalent redox centers in the same molecule depends not only on the nature of the bridging ligand but also on the geometrical arrangement of the redox centers.

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.

Iron in Oxidative and Reduction Processes in Marine Sands

The iron content and the ratio of bivalent to total iron in the labile acid-soluble fraction of iron were studied in sublittoral sands of Vostok Bay, Sea of Japan. Iron oxidation and reduction rates in sand sediment were measured in the laboratory. Trivalent iron almost always prevailed in the acid-extractable fraction in the natural environment. The most oxidized state of iron occurred in spring and was associated with a low water temperature and a high seawater oxygen content; the most reduced iron was found in fall during periods of low hydrodynamics and low oxygen concentration. In sand, iron is mainly reduced by bacteria; this process is slow and can be inhibited by adding chloroform. Oxidation of iron is mainly a chemical process and cannot be stopped by chloroform. In sand, the content of redox equivalents such as trivalent iron is much greater than in dissolved oxygen of pore water. It is assumed that labile iron in sands acts as a redox buffer, is oxidized by dissolved pore water oxygen at the periods of advective mixing, and is slowly reduced by benthic bacteria during anaerobic conditions.

The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox shuttle in Saccharomyces cerevisiae.

NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly, NDI1 is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogenases. Deletion of ADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures. Also, an ndi1Delta mutant was capable of respiratory growth under these conditions. However, when both ADH3 and NDI1 were deleted, metabolism became respirofermentative, indicating that the ethanol-acetaldehyde shuttle is essential for respiratory growth of the ndi1 delta mutant. In anaerobic batch cultures, the maximum specific growth rate of the adh3 delta mutant (0.22 h(-1)) was substantially reduced compared to that of the wild-type strain (0.33 h(-1)). This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions. Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in the adh3 delta ndi1 delta mutant, contributing to residual redox-shuttle activity in this strain.

Enantiomeric discrimination of Ru-substrates by cytochrome P450 cam

Molecules with photosensitizers attached to substrates (Wilker et al., Angew. Chem. Int. Ed. 38 (1999) 90–92) or cofactors (Hamachi et al., J. Am. Chem. Soc. 121 (1999) 5500–5506) can rapidly deliver redox equivalents to buried active sites. The structure of cytochrome P450 cam (P450) co-crystallized with a prototypal sensitizer-substrate, [Ru-C 9-Ad]Cl 2, has been determined (Dmochowski et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12987–12990); and, in separate UV–vis absorption and time-resolved luminescence experiments, the binding of the Λ and Δ enantiomers of Ru-C 9-Ad to P450 has been measured. The results, K D(Δ/Λ)∼2, indicate that the bipyridyl ligands of the Λ isomer interact more favorably with hydrophobic residues at the entrance to the substrate channel. We conclude that enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates.

Voltammetric behavior of poly(amidoamine) dendrimers containing nitroxyl radical end groups

A series of fully functionalized poly(amidoamine) dendrimers with 4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl end groups were prepared. Cyclic voltammetric studies indicated that the pendant nitroxyl radical end groups are non-interacting electrochemically equivalent redox centers, which are oxidizable at the same potential. The results of controlled-potential electrolysis of the dendrimers showed that all the nitroxyl radical groups at the periphery of the dendrimers are accessible to the electrode surface for electron transfer.