Internal Electron Transfer Research Articles

Unusual Multi-Step Sequential AuIII/AuII Processes of Gold(III) Quinoxalinoporphyrins in Acidic Non-Aqueous Media

The electrochemistry of gold(III) mono- and bis-quinoxalinoporphyrins was examined in CH(2)Cl(2) or PhCN containing 0.1 M tetra-n-butylammonium perchlorate (TBAP) before and after the addition of trifluoroacetic acid to solution. The investigated porphyrins are represented as Au(PQ)PF(6) and Au(QPQ)PF(6), where P is the dianion of the 5,10,15,20-tetrakis(3,5-di-tert-butylphenyl)porphyrin and Q is a quinoxaline group fused to a β,β'-pyrrolic position of the porphyrin macrocycle; in Au(QPQ)PF(6) there is a linear arrangement where the quinoxalines are fused to pyrrolic positions that are opposite each other. The porphyrin without the fused quinoxaline groups, Au(P)PF(6), was also investigated under the same solution conditions. In the absence of acid, all three gold(III) porphyrins undergo a single reversible Au(III)/Au(II) process leading to the formation of a Au(II) porphyrin which can be further reduced at more negative potentials to give stepwise the Au(II) porphyrin π-anion radical and dianion, respectively. However, in the presence of acid, the initial Au(III)/Au(II) processes of Au(PQ)PF(6) and Au(QPQ)PF(6) are followed by an internal electron transfer and protonation to regenerate new Au(III) porphyrins assigned as Au(III)(PQH)(+) and Au(III)(QPQH)(+). Both protonated gold(III) quinoxalinoporphyrins then undergo a second Au(III)/Au(II) process at more negative potentials. The electrogenerated monoprotonated monoquinoxalinoporphyrin, Au(II)(PQH), is then further reduced to its π-anion radical and dianion forms, but this is not the case for the monoprotonated bis-quinoxalinoporphyrin, Au(II)(QPQH), which accepts a second proton and is rapidly converted to Au(III)(HQPQH)(+) before undergoing a third Au(III)/Au(II) process to produce Au(II)(HQPQH) as a final product. Thus, Au(P)PF(6) undergoes one metal-centered reduction while Au(PQ)PF(6) and Au(QPQ)PF(6) exhibit two and three Au(III)/Au(II) processes, respectively. These unusual multistep sequential Au(III)/Au(II) processes were monitored by thin-layer spectroelectrochemistry and a reduction/oxidation mechanism for Au(PQ)PF(6) and Au(QPQ)PF(6) in acidic media is proposed.

Reorganization Energy for Internal Electron Transfer in Multicopper Oxidases

We have calculated the reorganization energy for the intramolecular electron transfer between the reduced type 1 copper site and the peroxy intermediate of the trinuclear cluster in the multicopper oxidase CueO. The calculations are performed at the combined quantum mechanics and molecular mechanics (QM/MM) level, based on molecular dynamics simulations with tailored potentials for the two copper sites. We obtain a reorganization energy of 91-133 kJ/mol, depending on the theoretical treatment. The two Cu sites contribute by 12 and 22 kJ/mol to this energy, whereas the solvent contribution is 34 kJ/mol. The rest comes from the protein, involving small contributions from many residues. We have also estimated the energy difference between the two electron-transfer states and show that the reduction of the peroxy intermediate is exergonic by 43-87 kJ/mol, depending on the theoretical method. Both the solvent and the protein contribute to this energy difference, especially charged residues close to the two Cu sites. We compare these estimates with energies obtained from QM/MM optimizations and QM calculations in a vacuum and discuss differences between the results obtained at various levels of theory.

Intramolecular electron transfer in laccases

Rate constants and activation parameters have been determined for the internal electron transfer from type 1 (T1) to type 3 (T3) copper ions in laccase from both the fungus Trametes hirsuta and the lacquer tree Rhus vernicifera, using the pulse radiolysis method. The rate constant at 298 K and the enthalpy and entropy of activation were 25 ± 1 s(-1), 39.7 ± 5.0 kJ·mol(-1) and -87 ± 9 J·mol(-1) ·K(-1) for the fungal enzyme and 1.1 ± 0.1 s(-1), 9.8 ± 0.2 kJ·mol(-1) and -211 ± 3 J·mol(-1) ·K(-1) for the tree enzyme. The initial reduction of the T1 site by pulse radiolytically produced radicals was direct in the case of T. hirsuta laccase, but occured indirectly via a disulfide radical in R. vernicifera. The equilibrium constant that characterizes the electron transfer from T1 to T3 copper ions was 0.4 for T. hirsuta laccase and 1.5 for R. vernicifera laccase, leading to full reduction of the T1 site occurring at 2.9 ± 0.2 electron equivalents for T. hirsuta and 4 electron equivalents for R. vernicifera laccase. These results were compared with each other and with those for the same process in other multicopper oxidases, ascorbate oxidase and Streptomyces coelicolor laccase, using available structural information and electron transfer theory.

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm(-1) range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(-), and 3,607(+) cm(-1). A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D(2)O and H(2)(18)O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.

Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase

CPR (NADPH-cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.

Anchoring of 1,8-naphthalimide derivative into mesoporous MCM-41 molecular sieves

The fluorescent dye molecules, 4-piperidine-1,8-naphthalimide, were successfully fixed into the amino modified pore channel of mesoporous MCM-41 type materials by in situ reaction of 4-piperidinyl-1,8-naphthalic anhydride with the amino group. The formation of amide bonds on the pore surface was verified by infrared spectra. The maximum fluorescence emission peak of this hybrid material has a red shift of 13 nm compared to that of a naphthalimide derivative in ethanol solution. Moreover, the fluorescence intensity of dye molecules grafted into Ce-doped MCM-41 is higher than that in pure silica MCM-41. This phenomenon is attributed to the inhibited internal electron transfer from piperidine to naphthalimide groups by Ce4+, thus improving the fluorescence intensity of the naphthalimide group. The unique fluorescence behavior of the 1,8-naphthalimide derivative doped hybrid mesoporous material makes it a good candidate for the metal ions microdetection.

Cloning, expression, and physicochemical characterization of a new diheme cytochrome c from Shewanella baltica OS155

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV-vis, magnetic circular dichroism, and (1)H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pK (a) values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of -0.144 and -0.257V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°'.

Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-dependent 2-Oxoacid Oxidoreductase That Enables Anaerobic Growth on Oxalate

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.

Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X-ray Crystallography

Schistosomiasis is the second most widespread human parasitic disease. It is principally treated with one drug, praziquantel, that is administered to 100 million people each year; less sensitive strains of schistosomes are emerging. One of the most appealing drug targets against schistosomiasis is thioredoxin glutathione reductase (TGR). This natural chimeric enzyme is a peculiar fusion of a glutaredoxin domain with a thioredoxin selenocysteine (U)-containing reductase domain. Selenocysteine is located on a flexible C-terminal arm that is usually disordered in the available structures of the protein and is essential for the full catalytic activity of TGR. In this study, we dissect the catalytic cycle of Schistosoma mansoni TGR by structural and functional analysis of the U597C mutant. The crystallographic data presented herein include the following: the oxidized form (at 1.9 Å resolution); the NADPH- and GSH-bound forms (2.3 and 1.9 Å, respectively); and a different crystal form of the (partially) reduced enzyme (3.1 Å), showing the physiological dimer and the entire C terminus of one subunit. Whenever possible, we determined the rate constants for the interconversion between the different oxidation states of TGR by kinetic methods. By combining the crystallographic analysis with computer modeling, we were able to throw further light on the mechanism of action of S. mansoni TGR. In particular, we hereby propose the putative functionally relevant conformational change of the C terminus after the transfer of reducing equivalents from NADPH to the redox sites of the enzyme.

ChemInform Abstract: Endohedral Effect in Inclusion Complexes of the C60 Cluster

An approximate theory of bonding in endohedral complexes that are formed by atoms, ions, and molecules trapped inside the C60 cage is formulated. It is found that the electrostatic potential at the cage center (the endohedral potential) determines such electronic properties of the endohedral complexes as the complexation energy, ionization potentials, and the stability with respect to internal electron transfer. The origins of the endohedral effect, i.e., the positive valuedness of the endohedral potential, are investigated with the help of the Thomas–Fermi theory. The theoretical predictions are demonstrated to be in good agreement with the numerical results of ab initio electronic structure calculations.

Geometric structures and electron affinities of chlorine-doped silicon clusters

The electronic and geometrical structures of the ground and possible low-lying isomers of Si n Cl and Si n Cl– (n = 1–6) were investigated at the B3LYP/6-31G(d) and B3LYP/6-311+G(d) levels. The natural populations and natural electron configurations are presented and discussed for the most stable structures. The impact of internal electron transfer between the (Si n ) subsystem and the Cl atom of Si n Cl− (Si n Cl) clusters is investigated. Furthermore, the three different types of electron affinities for all chlorine-doped silicon clusters are reported, and the dissociation energies are also evaluated for both series using Møller–Plesset and coupled cluster methods.

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) (2NO + 2H(+) + 2e(-) -->N(2)O + H(2)O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b(3), and a non-heme iron Fe(B). Here, we have studied the reaction between fully reduced NOR and NO using the "flow-flash" technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b(3) occurs with a time constant of approximately 1 micros at 1.5 mM NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe(B) before ligation to heme b(3). Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s(-1) and 3 s(-1) at 1.5 mM NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s(-1) and 12 s(-1) at 75 microM NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.

Potentially Deadly Carcinogenic Chromium Redox Cycle Involving Peroxochromium(IV) and Glutathione

Peroxochromium(IV) complexes are putative DNA-damaging and mutagenic agents in chromium(VI)-mediated carcinogenesis. The reaction between aquaethylenediaminebis(peroxo)chromium(IV) and glutathione at neutral pH exhibits a cyclic redox process displaying a persistent recycling of Cr(IV) and Cr(VI) with the intervention of chromium(V) intermediates. The coordination by a glutathione molecule triggers an autooxidation of the Cr(IV)-peroxo complex to Cr(VI) via an internal electron-transfer process followed by reduction to Cr(IV) via metastable chromium(V) intermediates. The cycle is repeated by the second peroxo species. The Cr(V) and -(IV) intermediates have been characterized as mono- and bisglutathionato complexes with or without a coordinated peroxo moiety. These intermediates are capable of damaging DNA, as evidenced by single strand breaks and DNA oxidation. The implication here is that the potential for a persistent, if not perpetual, deadly chromium carcinogenic cycle exists in the cellular milieu through the assistance of molecular oxygen and glutathione.

Nature of the Energy Landscape for Gated Electron Transfer in a Dynamic Redox Protein

Conformational control limits most electron transfer (ET) reactions in biology, but we lack general insight into the extent of conformational space explored, and specifically the properties of the associated energy landscape. Here we unite electron-electron double resonance (ELDOR) studies of the diradical (disemiquinoid) form of human cytochrome P450 reductase (CPR), a nicotinamide adenine phosphate dinucleotide (NADPH)-linked diflavin oxidoreductase required for P450 enzyme reduction, with functional studies of internal ET to gain new insight into the extent and properties of the energy landscape for conformationally controlled ET. We have identified multiple conformations of disemiquinoid CPR, which point to a rugged energy landscape for conformational sampling consistent with functional analysis of ET using high-pressure stopped-flow, solvent, and temperature perturbation studies. Crystal structures of CPR have identified discrete "closed" and "open" states, but we emphasize the importance of a continuum of conformational states across the energy landscape. Within the landscape more closed states that favor internal ET are formed by nucleotide binding. Open states that enable P450 enzymes to gain access to electrons located in the FMN-domain are favored in the absence of bound coenzyme. The extent and nature of energy landscapes are therefore accessible through the integration of ELDOR spectroscopy with functional studies. We suggest this is a general approach that can be used to gain new insight into energy landscapes for biological ET mediated by conformational sampling mechanisms.

PHOTO-REDUCTION KINETICS OF MnO<sub>2</sub> IN AQUATIC ENVIRONMENTS CONTAINING HUMIC ACIDS

The kinetics of photo-reduction of manganese(IV) oxide, MnO2 in aqueous medium, in the presence of humic acid has been investigated, including the possibility for the involvement of such radicals in the reduction. Results showed that in aqueous medium, the efficiency of the photo-reduction of MnO2 producing a soluble ionic Mn(II) was governed strongly by an internal electron transfer within an intermediate of HA-MnO2 anion complex, resulted in the chemical adsorption of humic acid on MnO2 surface. It was observed that under solar radiation and atmosphere containing oxygen, the rate of MnO2 reduction increased considerably, by factors of about 46 and 2 respectively. Moreover, there was evidence that some radicals certainly played a role in the photo-reduction of MnO2, since radical scavenger addition significantly decreased the reduction efficiency of MnO2. In the present of an excess of propanol-2, however, which was expected to remove all OH radicals present, only about 60 % decrease in reduction efficiency were observed, suggesting that radicals other than OH radical are involved in the reduction of MnO2. It was observed further that the presence of iron(III) in MnO2 up to 1 % (w/w) did not alter the reduction efficiency of MnO2. Keywords: MnO2 kinetics, photo-reduction, aquatic, humic acids

Mechanistic Study of Amine to Imine Oxidation in a Dinuclear Cu(II) Complex Containing an Octaaza Dinucleating Ligand

Density functional theory (DFT) calculations have been carried out to elucidate the mechanism of self-oxidation of a Cu(II) complex octaaza dinucleating macrocyclic ligand. The reaction is bimolecular and spontaneous, in which amine groups of one macrocycle are oxidized and the Cu(II) centers of a second macrocylic complex are reduced. No additional oxidation or external base agents are required. DFT calculations predict the reaction to proceed via a two-step mechanism, in which the first step is proton transfer between two reactant complexes. This is followed by a second transfer step in which an electron and proton are transferred together between the two complexes. Concurrent with this external transfer there is also an internal electron transfer in which the ligand reduces the metal center to give the imine product bound to Cu(I). The complexity of this final step differs from the generally accepted mechanisms for transition metal catalyzed amine to imine oxidation in which protons and electrons are transferred individually.

Site−Site Interactions Enhances Intramolecular Electron Transfer in Streptomyces coelicolor laccase

Control of electron transfer rates, caused by intrinsic protein structural properties, is an intriguing feature of internal biological electron transfer (ET) reactions. The small laccase (SLAC) isolated from Streptomyces coelicolor has recently been shown to have structural and reactivity features distinct from those of other laccases. While other copper oxidases contain three cupredoxin domains, the SLAC 3D structure has recently been determined and shown to consist of only two, and a different reaction intermediate has been reported for it. It was therefore of particular interest to investigate the intramolecular ET between the type 1 and the trinuclear copper center in SLAC which is a crucial step in the catalytic cycle of the multicopper oxidases, leading to dioxygen reduction to water. This ET step was found to markedly depend on the reduction state of the enzyme, possibly reflecting site-site interactions so far not observed in other multicopper oxidases.

Voltage-Dependent Low-Field Bulk Resistivity in BaTiO3:Zn Ceramics

Ceramics of composition Ba(Ti1−xZnx)O3−x: 0.00003≤x≤0.01 exhibit modest semiconductivity, whose value is increased by up to two orders of magnitude under the action of a small dc bias voltage. The effect, which is not observed in undoped BaTiO3, occurs in both grain and grain boundary regions and is attributed to the nature of the defect structure, which contains highly polarized clusters. It is proposed that internal electron transfer within the clusters under the action of a dc bias voltage leads to a more conductive excited state, which gradually returns to the ground state when the dc bias is removed.

Control of Respiration by Cytochrome c Oxidase in Intact Cells

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.