Redox Communication Research Articles

Diversification of Quiescin sulfhydryl oxidase in a preserved framework for redox relay

BackgroundThe enzyme family Quiescin Sulfhydryl Oxidase (QSOX) is defined by the presence of an amino-terminal thioredoxin-fold (Trx) domain and a carboxy-terminal Erv family sulfhydryl oxidase domain. QSOX enzymes, which generate disulfide bonds and transfer them to substrate proteins, are present in a wide variety of eukaryotic species including metazoans and plants, but are absent from fungi. Plant and animal QSOXs differ in their active-site amino acid sequences and content of non-catalytic domains. The question arises, therefore, whether the Trx-Erv fusion has the same mechanistic significance in all QSOX enzymes, and whether shared features distinguish the functional domains of QSOX from other instances in which these domains occur independently. Through a study of QSOX phylogeny and an analysis of QSOX sequence diversity in light of recently determined three-dimensional structures, we sought insight into the origin and evolution of this multi-domain redox alliance.ResultsAn updated collection of QSOX enzymes was used to confirm and refine the differences in domain composition and active-site sequence motif patterns of QSOXs belonging to various eukaryotic phyla. Beyond the expected phylogenetic distinction of animal and plant QSOX enzymes, trees based on individual redox-active QSOX domains show a particular distinction of the Trx domain early in plant evolution. A comparison of QSOX domains with Trx and Erv domains from outside the QSOX family revealed several sequence and structural features that clearly differentiate QSOXs from other enzymes containing either of these domains. Notably, these features, present in QSOXs of various phyla, localize to the interface between the Trx and Erv domains observed in structures of QSOX that model interdomain redox communication.ConclusionsThe infrastructure for interdomain electron relay, previously identified for animal and parasite QSOXs, is found broadly across the QSOX family, including the plant enzymes. We conclude that the conserved three-dimensional framework of the QSOX catalytic domains accommodates lineage-specific differences and paralog diversification in the amino acid residues surrounding the redox-active cysteines. Our findings indicate that QSOX enzymes are characterized not just by the presence of the two defining domain folds but also by features that promote coordinated activity.

Electrochemistry of bilirubin oxidase and its use in preparation of a low cost enzymatic biofuel cell based on a renewable composite binder chitosan

Spherical carbon nanoparticles KetjenBlack (KB) with a high sorption capacity together with conductivity increasing single walled carbon nanotubes (CNTs) were “glued” together by chitosan for the preparation of a composite. A biocathode with bilirubin oxidase (BOD) adsorbed within the composite was characterised and its composition optimised. A renewable biopolymer chitosan present in the composite offered (1) pre-concentration of BOD within the matrix via electrostatic interactions, (2) favourable orientation of BOD for a direct electron transfer (DET) between BOD and the composite, (3) electrochemical visibility of 3 redox sites present in BOD, (4) low charge transfer resistance, (5) high proton conductivity and (6) low overpotential for oxygen reduction. Electrochemical investigation of BOD revealed interesting internal redox communication within the enzyme with some novel insights provided. At least one of tyrosines present in BOD seems to be involved in electron transfer route, as well. The composite used for the biocathode was directly applied for the preparation of a bioanode with fructose dehydrogenase immobilised, working in a DET mode of operation. Finally, integration of the biocathode and the bioanode into a biofuel cell operated in a reagentless and membraneless mode offered a power density of 50μWcm−2 at 300mV.

Sensing array of radically coupled genetic biopixels

While there has been significant progress in the development of engineering principles for synthetic biology, a substantial challenge is the construction of robust circuits in a cellular environment that is governed by noisy processes such as random bursts of transcription and translation. Such an environment leads to considerable intercellular variability in circuit behavior, which can hinder their functionality at the colony level. While quorum sensing is a promising design strategy for reducing variability through coordination across a cellular population, the length scales are limited by the diffusion time of the small molecule governing the intercellular communication. In this talk, I will discuss our recent progress in engineering the synchronization of thousands of oscillating colony “biopixels” over centimeter length scales through the use of redox signaling that is mediated by hydrogen peroxide vapor. We have used the redox communication to construct a frequency modulated biosensor by coupling the synchronized oscillators to the output of an arsenic sensitive promoter that modulates the frequency of colony‐level oscillations due to quorum sensing. For the detection of arsenic, we have developed thresholding and period modulation as sensing mechanisms. In the case of thresholding, oscillations arise when a pre‐determined low level of arsenic is present, while for period modulation the level of arsenic is proportional to the oscillatory frequency. In both cases, the coupling of thousands of colonies dramatically increases the signal to noise ratio and leads to the reliable detection of extremely low levels of arsenic. Given the repertoire of sensing capabilities of bacteria such as E. coli, the ability to coordinate their behavior over large length scales sets the stage for the construction of low cost genetic biosensors that are capable of detecting heavy metals and pathogens in the field.

Extracellular communication and signaling by thiol/disulfide redox states

Extracellular cysteine/cystine (Cys/CySS) and GSH/GSSG couples provide redox communication between organ systems. Both are highly regulated in healthy individuals. Cys and CySS are more abundant in human plasma, regulated by cell efflux and uptake mechanisms. Oxidized redox potentials (EhCySS and EhGSSG) and high CySS are associated with risk factors for cardiovascular disease (CVD), including aging, smoking, type 2 diabetes, obesity, and alcohol abuse. Oxidized EhCySS activates proinflammatory signaling through cell surface protein thiols, signals mitochondrial oxidant production, activates NF-κB and Nrf-2, and elevates proinflammatory cytokines, proapoptotic proteins, cell adhesion molecules and detoxification proteins. Reduced EhCySS stimulates cell proliferation and cell survival signaling in multiple cell types. The critical nature of these signaling mechanisms supports the use of plasma levels of Cys, CySS, and EhCySS as key indicators of systemic oxidative stress. Pharmacologic interventions to control or improve EhCySS may prove useful to prevent CVD and other age-associated disease processes.

Thiol Chemistry in Peroxidase Catalysis and Redox Signaling

The oxidation chemistry of thiols and disulfides of biologic relevance is described. The review focuses on the interaction and kinetics of hydrogen peroxide with low-molecular-weight thiols and protein thiols and, in particular, on sulfenic acid groups, which are recognized as key intermediates in several thiol oxidation processes. In particular, sulfenic and selenenic acids are formed during the catalytic cycle of peroxiredoxins and glutathione peroxidases, respectively. In turn, these enzymes are in close redox communication with the thioredoxin and glutathione systems, which are the major controllers of the thiol redox state. Oxidants formed in the cell originate from several different sources, but the major producers are NADPH oxidases and mitochondria. However, a different role of the oxygen species produced by these sources is apparent as oxidants derived from NADPH oxidase are involved mainly in signaling processes, whereas those produced by mitochondria induce cell death in pathways including also the thioredoxin system, presently considered an important target for cancer chemotherapy.

Identification of Acid-Base Catalytic Residues of High-Mr Thioredoxin Reductase from Plasmodium falciparum

High-M(r) thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.

Carbon nanotube-functionalized silicon surfaces with efficient redox communication

Sidewall functionalized multi-walled carbon nanotubes can be covalently bound parallel to a silicon surface via a self-assembled acid-terminated monolayer used as an organic molecular glue.

Ligand‐—Ligand Redox Interaction Through Some Metal‐Cluster Units

AbstractFor Abstract see ChemInform Abstract in Full Text.

Ligand–ligand redox interaction through some metal‐cluster units

The ligand-ligand redox interaction separated by di-, tri-, and hexanuclear cluster units is discussed in terms of the splitting of the redox waves (DeltaE(L)) and the comproportionation constants (Kcom(L)) of the ligand-based mixed valence state. Although two redox-active monodentate ligands in the mononuclear ruthenium(II) complexes (either cis or trans configurations) do not show appreciable splitting in their reduction waves, interestingly those separated by giant triruthenium and hexarhenium cluster units clearly show splitting. The molecular orbital considerations in the literature suggest that these units possess some pi-type molecular orbitals composed of metal dpi and possibly ligand ppi orbitals. Absence of the redox interactions in oxo-bridged diruthenium(II) and oxo-centered trirhodium(III) complexes where such pi molecular orbitals (including an antibonding one) are fully occupied, indicates the importance of empty pi* orbitals for the ligand redox communication.

Electrochemistry within molecules using ultrafast cyclic voltammetry

The basic principles allowing undistorted ultrafast cyclic voltammetry to be performed at ultramicroelectrodes are recalled and discussed as well as their experimental exploitation leading to the design of an ultrafast potentiostat allowing on-line ohmic drop compensation without distortions within the megavolts-per-second range of scan rates. The validity of the instrument and of the method are established by investigating the voltammetry of anthracene reduction up to scan rates of 2.5 MV s –1. Access to this range of ultrahigh scan rates allows nanosecond-time scale to be explored voltammetrically which offers obvious kinetic advantages by widening the mechanistic scope of voltammetry so that it encompasses now more than nine orders of magnitude. Yet, in this feature article, we are more interested in discussing another remarkable – but less promoted – advantage of ultrafast voltammetry. Indeed, scan rates in the megavolts- per-second range allow the creation of diffusion layers with a maximal extension of a few nanometers, viz, which are comparable to the size of chemical and biological molecules. In other words, this allows the in-situ and direct monitoring of redox communication within molecules that are attached to an ultramicroelectrode surface. The experimental validity and the interest of this concept are presented on the basis of two typical examples. One deals with the rate of redox commutation within a self-assembled monolayer of organometallic molecules bearing a redox terminal group, [Os(bpy) 2Cl(py–B)] +, adsorbed onto a platinum surface through the pyridyl moiety of their ligated bridge (–B = –(CH 2) 2–py). The second application concerns the monitoring of the time progression of electron-hopping diffusion through electron exchange between 64 Ru II/III(tpy) 2 redox groups distributed on a fourth-generation PAMAM dendrimer molecule adsorbed onto an ultramicroelectrode. Besides the measurement of the rate of communication between adjacent ruthenium centers within one dendrimer, the method is extremely informative about the topology of the spatial arrangement of the 64 redox centers, so that it can be used as a molecular microtome with a resolution better than that of STM. To cite this article: C. Amatore et al., C. R. Chimie 6 (2003) 000–000.

Bioelectrocatalysis by redox enzymes at modified electrodes

Self-assembled monolayers of thiolated compounds are used as promoters for protein–electrode reactions. They provide an anchor group based on thiol chemisorptions and also a functional group for effective interaction with the protein. These interactions are often governed by electrostatic attraction. For example, for positively charged proteins, such as cytochrome c and the selenoprotein glutathione peroxidase, mercaptoalkanoic acids have been used. Clay modification of the electrode surface has been found to facilitate the heterogeneous electron transfer process for heme proteins, e.g. cytochrome c, cytochrome P450 and myoglobin. Interestingly, nucleic acids at carbon electrodes and thiol-modified double stranded oligonucleotides act as promoters of the redox communication to proteins, whereas the mechanism is still subject to controversy interpretations. By interacting the protein immobilised at the electrode with species in solution, signal chains have been constructed. The interaction can result in a simple co-ordination or redox reaction, depending on the nature of the reaction partners. For analytical purposes, e.g. biosensors, the electrochemical redox conversion of the immobilised protein is evaluated.

Investigation of Electronic Communication and Guest Inclusion Using Photoluminescent Macrocyclic Receptors with Ru(II) Centers and Ph(2)P-C identical withC-C identical withC-PPh(2) Spacers This work was supported by the US National Science Foundation CAREER Award (CHE-9733546).

An enhanced photoluminescence is observed upon addition of organic guests to the trimeric macrocycle (see picture; tpy=2,2′:6′,2″-terpyridine). The use of a short alkyne bridge has enabled the rigid trimer and the smaller dimer to be prepared with controllable cavity sizes. A better redox communication between the Ru centers in the dimer is also observed.

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli.

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.

Inhibition of Rat Liver Mitochondrial Permeability Transition by Respiratory Substrates

The mitochondrial inner membrane can undergo a permeability increase known as "permeability transition" elicited by Ca2+ and several other inducing agents. In general, the condition of oxidative stress acts as an inducer, at variance with antioxidants and reducing agents that inhibit the permeability transition. The action of mitochondrial respiratory substrates in preventing the permeability transition induced by Ca2+ and phosphate was examined; pyruvate, isocitrate, and glutamate proved to be particularly effective. The effect of substrates was evident also in the presence of an uncoupler, and, in addition, they were able to counteract the swelling stimulated by acetoacetate and tert-butylhydroperoxide. In the presence of various pyridine nucleotide-dependent substrates, mitochondria are able to reduce the disulfide 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) to an extent far larger than that calculated from the theoretical amount of total mitochondrial thiol groups, indicating the occurrence of a catalytic system. Similarly, the enzymes of the mitochondrial matrix in the presence of either NADH or NADPH are able to reduce DTNB. The results are discussed considering the existence of a close redox communication between pyridine nucleotides and membrane thiol groups, possibly mediated by dithiols such as thioredoxin and lipoic acid.

Control of electrochemical processes by photoisomerizable spiropyran monolayers immobilized onto Au electrodes: amperometric transduction of optical signals

Photoisomerizable self-assembled spiropyran (SP) monolayers on Au electrodes provide a means of transducing optical signals recorded by the monolayers as amperometric responses. An SP-modified monolayer on a Au electrode photostimulated the redox communication of pyrroloquinolinoquinone (PQQ) with the electrode interface. The electrical communication of PQQ with the SP monolayer state generated by visible light irradiation (λ > 475 nm) is inefficient. However, effective electrical communication is attained between PQQ and the protonated merocyanine monolayer electrode ((MRH)+ electrode) generated by illumination (360 nm < λ < 400 nm). Reversible photoisomerization of the monolayer between the SP and (MRH)+ states is transduced by cyclic amperometric responses. A mixed PQQ + SP monolayer on a Au electrode provides an active electrode for the photostimulated oxidation of NAD(P)H cofactors in the presence of Ca2+ ions. While the PQQ + SP monolayer electrode exhibits effective electrocatalytic properties towards the oxidation of NAD(P)H, the PQQ + (MRH)+ monolayer electrode state reveals poor electrocatalytic properties towards oxidation of the cofactors. Reversible photoisomerization of the monolayer across the PQQ + SP and PQQ + MRH)+ states provides a means of transducing the recorded optical signals by means of the electrocatalyzed oxidation of the cofactors as light-controlled amperometric responses.