Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to γ-linolenic acid in rat liver microsomes

The effects of antibodies against microsomal electron-transport components on the in vitro activity of Δ⁶-desaturation of linoleic acid to γ-linolenic acid have been studied in intact microsomal membranes of rat liver. Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.87 mM) served as electron donors, and effectively prompted the Δ⁶-desaturase activities with yields of about 1.1 to 1.3 nmol per mg of protein in 10 min. Of the two antibodies studied under the same in vitro conditions, i.e., rabbit antisera preparations against rat liver microsomal hydrophilic parts of cytochrome b₅ and NADPH-cytochrome c reductase, only the antibody against cytochrome b₅ demonstrated a marked ability to inhibit the Δ⁶-desaturase activity. This evidence supports a participation of cytochrome b₅ in the Δ⁶-desaturation of linoleic acid and suggests a pathway analogous to the Δ⁹-desaturation of stearyl-CoA.     

Electrochemical study of the intermolecular electron transfer to Pseudomonas aeruginosa cytochrome cd1 nitrite reductase

The kinetics of electron transfer reaction between cytochrome cd₁ nitrite reductase (NiR) from Pseudomonas aeruginosa and various physiological/non physiological redox partners was investigated using cyclic voltammetry at the pyrolytic graphite electrode. While NiR did not exchange electron with the electrode, cytochrome c₅₅₁ and azurin, both from Ps. aeruginosa, behaved as fast electrochemical systems. The intermolecular electron transfers between NiR and cytochrome c₅₅₁ or azurin as electron shuttles, in the presence of nitrite, were studied. Second order rate constants of 2×10⁶ and 1.4×10⁵ M⁻¹ s⁻¹ are calculated for cytochrome c₅₅₁ and azurin, respectively. The dependence of the second-order rate constant on ionic strength and pH is discussed. Finally, the effect of the global charge of the electron shuttles was explored using differently charged species (proteins or small ions). The experimental results suggest involvement of polar interactions as well as of hydrophobic contacts in the protein recognition prior to the intermolecular electron transfer. As the cross-reaction between Ps. nautica cytochrome c₅₅₂ and Ps. aeruginosa NiR was shown to be as efficient as the catalytic reaction involving the physiological partners, it is concluded to a ‘pseudo-specificity’ in the recognition between NiR and the electron donor.     

The influence of Glu44 and Glu56 of cytochrome b5 on the protein structure and interaction with cytochrome c

The gene encoding trypsin-solubilized bovine liver microsomalcytochrome b₅ (82 residues in length) has been mutated, in whichthe codons of Glu44 and Glu56 were changed to those of Ala.The mutated genes were expressed in Escherichia coli successfullyand three mutant proteins (E44A, E56A and E44/56A) were obtained.The UV-visible, CD and ¹H NMR spectra of proteins have beenstudied. The results show that the mutagenesis at surface residuesdoes not alter the secondary and tertiary structures of cytochromeb₅ significantly. The interactions between recombinant cytochromeb₅ and its mutants with cytochrome c were studied by using opticaldifference spectra. The results demonstrated that both Glu44and Glu56 of cytochrome b₅ participate in the formation of acomplex between cytochrome b₅ and cytochrome c.     

Desaturases fused to their electron donor

Fatty acid desaturations in the carboxy‐terminal segment from C1—C10 are catalyzed in many, but not in all cases, by desaturase enzymes which are fused to their electron donor cytochrome b₅. Several of these enzymes (“front‐end desaturases”) from a wide variety of organisms have been cloned and functionally expressed for proof of regio‐, stereo‐ and chain length‐selectivity. In most cases the actual status of the substrate chain, whether coenzyme A thioester or component of a membrane lipid, is not known. The cytochrome b₅ domain is located N‐terminally, internally or C‐terminally. Compared to the free cytochrome b₅ , the fused domains show a significant reduction of acidic amino acid residues on the surface of the four helices enclosing the heme group. It is discussed how this may contribute to hydrophobic domain pairing required for interdomain electron transport. This is in contrast to the mode of interaction of free cytochrome b₅ with its partners, which is governed by electrostatic charge pairing. A look at crystallized or computer‐simulated models involving fused or free cytochrome b₅ helps to outline the problems encountered by optimizing the docking of partners and the exchange of electrons between domains of different degrees of mobility.     

Electron Transfer Pathways in Cholesterol Synthesis

Cholesterol synthesis in the endoplasmic reticulum requires electron input at multiple steps and utilizes both NADH and NADPH as the electron source. Four enzymes catalyzing five steps in the pathway require electron input: squalene monooxygenase, lanosterol demethylase, sterol 4α‐methyl oxidase, and sterol C5‐desaturase. The electron‐donor proteins for these enzymes include cytochrome P450 reductase and the cytochrome b₅ pathway. Here I review the evidence for electron donor protein requirements with these enzymes, the evidence for additional electron donor pathways, and the effect of deletion of these redox enzymes on cholesterol and lipid metabolism.     

Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced Q_B and the S₂/S₃ states of the water-splitting machinery. However, electron transfer from Q_A to Q_B, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c₅₅₀ subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c₅₅₀ under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c₅₅₀, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.     

Superoxide Reductase: Different Interaction Modes with its Two Redox Partners

Anaerobic organisms have molecular systems to detoxify reactive oxygen species when transiently exposed to oxygen. One of these systems is superoxide reductase, which reduces O₂^.? to H₂O₂ without production of molecular oxygen. In order to complete the reduction of superoxide anion, superoxide reductase requires an electron, delivered by its redox partners, which in Desulfovibrio gigas are rubredoxin and/or desulforedoxin. In this work, we characterized the interaction of Desulfovibrio gigas superoxide reductase with both electron donors by using steady‐state kinetics, 2D NMR titrations, and backbone relaxation measurements. The rubredoxin surface involved in the electron transfer complex with superoxide reductase comprises the solvent‐exposed hydrophobic residues in the vicinity of its metal center (Cys9, Gly10, Cys42, Gly43, and Ala44), and a K_d of 3 μM at 59 mM ionic strength was estimated by NMR. The ionic strength dependence of superoxide‐mediated rubredoxin oxidation by superoxide reductase has a maximum k_app of (37±12) min^?1 at 157 mM . Relative to the electron donor desulforedoxin, its complex with superoxide reductase was not detected by chemical shift perturbation, though this protein is able to transfer electrons to superoxide reductase with a maximum k_app of (31±7) min^?1 at an ionic strength of 57 mM . Competition experiments using steady‐state kinetics and NMR spectroscopy (backbone relaxation measurements and use of a paramagnetic relaxation enhancement probe) with Fe‐desulforedoxin in the presence of ¹⁵N‐Zn‐rubredoxin showed that these two electron donors compete for the same site on the enzyme surface, as shown in the model structure of the complex generated by using restrained molecular docking calculations. These combined strategies indicate that the two small electron donors bind in different manners, with the desulforedoxin complex being a short lived electron transfer complex or more dynamic, with many equivalent kinetically competent orientations.     

Facile Oxidation of Leucomethylene Blue and Dihydroflavins by Artemisinins: Relationship with Flavoenzyme Function and Antimalarial Mechanism of Action

The antimalarial drug methylene blue (MB) affects the redox behaviour of parasite flavin‐dependent disulfide reductases such as glutathione reductase (GR) that control oxidative stress in the malaria parasite. The reduced flavin adenine dinucleotide cofactor FADH₂ initiates reduction to leucomethylene blue (LMB), which is oxidised by oxygen to generate reactive oxygen species (ROS) and MB. MB then acts as a subversive substrate for NADPH normally required to regenerate FADH₂ for enzyme function. The synergism between MB and the peroxidic antimalarial artemisinin derivative artesunate suggests that artemisinins have a complementary mode of action. We find that artemisinins are transformed by LMB generated from MB and ascorbic acid (AA) or N‐benzyldihydronicotinamide (BNAH) in situ in aqueous buffer at physiological pH into single electron transfer (SET) rearrangement products or two‐electron reduction products, the latter of which dominates with BNAH. Neither AA nor BNAH alone affects the artemisinins. The AA–MB SET reactions are enhanced under aerobic conditions, and the major products obtained here are structurally closely related to one such product already reported to form in an intracellular medium. A ketyl arising via SET with the artemisinin is invoked to explain their formation. Dihydroflavins generated from riboflavin (RF) and FAD by pretreatment with sodium dithionite are rapidly oxidised by artemisinin to the parent flavins. When catalytic amounts of RF, FAD, and other flavins are reduced in situ by excess BNAH or NAD(P)H in the presence of the artemisinins in the aqueous buffer, they are rapidly oxidised to the parent flavins with concomitant formation of two‐electron reduction products from the artemisinins; regeneration of the reduced flavin by excess reductant maintains a catalytic cycle until the artemisinin is consumed. In preliminary experiments, we show that NADPH consumption in yeast GR with redox behaviour similar to that of parasite GR is enhanced by artemisinins, especially under aerobic conditions. Recombinant human GR is not affected. Artemisinins thus may act as antimalarial drugs by perturbing the redox balance within the malaria parasite, both by oxidising FADH₂ in parasite GR or other parasite flavoenzymes, and by initiating autoxidation of the dihydroflavin by oxygen with generation of ROS. Reduction of the artemisinin is proposed to occur via hydride transfer from LMB or the dihydroflavin to O1 of the peroxide. This hitherto unrecorded reactivity profile conforms with known structure–activity relationships of artemisinins, is consistent with their known ability to generate ROS in vivo, and explains the synergism between artemisinins and redox‐active antimalarial drugs such as MB and doxorubicin. As the artemisinins appear to be relatively inert towards human GR, a putative model that accounts for the selective potency of artemisinins towards the malaria parasite also becomes apparent. Decisively, ferrous iron or carbon‐centered free radicals cannot be involved, and the reactivity described herein reconciles disparate observations that are incompatible with the ferrous iron–carbon radical hypothesis for antimalarial mechanism of action. Finally, the urgent enquiry into the emerging resistance of the malaria parasite to artemisinins may now in one part address the possibilities either of structural changes taking place in parasite flavoenzymes that render the flavin cofactor less accessible to artemisinins or of an enhancement in the ability to use intra‐erythrocytic human disulfide reductases required for maintenance of parasite redox balance.     

Influence of ionic liquids on the direct electrochemistry of glucose oxidase entrapped in nanogold-N,N-dimethylformamide-ionic liquid composite film

Glucose oxidase (GOD) immobilized in nanogold particles (NAs)-N,N-dimethylformamide (DMF) composite film on glassy carbon (GC) electrode exhibits a pair of quasi-reversible and unstable peaks due to the redox of flavin adenine dinucleotide (FAD) of GOD. When ionic liquids (ILs) 1-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF₄) or trihexyltetradecylphosphorium bis (trifluoromethylsulfony) (P_666,14 NTf₂) is introduced in the film, the peaks become small. But ILs 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆) and 1-octyl-3-methylimidazolium hexafluorophate (OMIMPF₆) make the peaks large and stable. In different composite films the formal potential (E⁰′) of GOD is different. UV-vis spectra show that the GOD dispersed in these films almost retains its native structure and there are weak interactions between ILs and GOD. Electrochemical impedance spectra display that NAs can promote the electron transfer between FAD and GC electrode; and ILs can affect the electron transfer through interacting with GOD. The thermal stability of GOD entrapped in NAs-DMF-ILs composite films is also influenced by ILs, and it follows such order as: in NAs-DMF-OMIMPF₆ > in NAs-DMF-BMIMPF₆ ≈ in NAs-DMF-BMIMBF₄ > in NAs-DMF. In addition, GOD immobilized in NAs-DMF-OMIMPF₆ and NAs-DMF-BMIMPF₆ films shows good catalytic activity to the oxidation of glucose. The I_max of H₂O₂ and the apparent K_m (Michaelis-Menten constant) for the enzymatic reaction are calculated.     

Flavin‐N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme

The flavin‐dependent enzyme 2‐haloacrylate hydratase (2‐HAH) catalyzes the conversion of 2‐chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2‐HAH was shown to be monomeric in solution and contained a non‐covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox‐neutral, 2‐HAH was active only in the reduced state. A covalent flavin‐substrate intermediate, consistent with the flavin‐acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small‐angle X‐ray scattering was consistent with 2‐HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2‐HAH as a novel noncanonical flavoenzyme.     

Effects of the gold nanoparticles including different thiol functional groups on the performances of glucose-oxidase-based glucose sensing devices

Thiol-based self-assembled anchor linked to glucose oxidase (GOx) and gold nanoparticle (GNP) cluster is suggested to enhance the performance of glucose biosensor. By the adoption of thiol-based anchors, the activity of biocatalyst consisting of GOx, GNP, polyethyleneimine (PEI) and carbon nanotube (CNT) is improved because they play a crucial role in preventing the leaching out of GOx. They also promote electron collection and transfer, and this is due to a strong hydrophobic interaction between the active site of GOx and the aromatic ring of anchor, while the effect is optimized with the use of thiophenol anchor due to its simple configuration. Based on that, it is quantified that by the adoption of thiophenol as anchor, the current density of flavin adenine dinucleotide (FAD) redox reaction increases about 42%, electron transfer rate constant (k_s) is 9.1±0.1 s^?1 and the value is 26% higher than that of catalyst that does not use the anchor structure.     

Cloning,Expression and Purification of Cindoxin,an Unusual Fmn‐Containing Cytochrome P450 Redox Partner

Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450_cin (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH‐dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6<pH<8; this enabled the quinone/semiquinone (E₁) and semiquinone/hydroquinone (E₂) redox potentials to be determined. Our results show that cindoxin might be somewhat different to other flavodoxins that interact with P450s, in which generally only one couple is important. Both redox states of cindoxin could be catalytically relevant. A catalytically active system was reconstituted in vitro with E. coli flavodoxin reductase (Fpr) acting as the terminal redox partner in the absence of CdR. Our results show that Cdx and Fpr support regio‐ and stereoselective P450_cin‐catalysed cineole oxidation to (1R)‐6β‐hydroxycineole with turnover rates up to 1500 min^?1. This system is tightly coupled with 80 % of NADPH reducing equivalents funnelled into substrate oxidation.     

A Clickable Photosystem I,Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration**

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b₆f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP⁺ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI−Fd and PSI−Fd−FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI−PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7−PSI was coupled with the E7−Fd or E7−Fd−FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.     

Catalytic and Spectroscopic Properties of the Halotolerant Soluble Methane Monooxygenase Reductase from Methylomonas methanica MC09

The soluble methane monooxygenase receives electrons from NADH via its reductase MmoC for oxidation of methane, which is itself an attractive C1 building block for a future bioeconomy. Herein, we present biochemical and spectroscopic insights into the reductase from the marine methanotroph Methylomonas methanica MC09. The presence of a flavin adenine dinucleotide (FAD) and [2Fe2S] cluster as its prosthetic group were revealed by reconstitution experiments, iron determination and electron paramagnetic resonance spectroscopy. As a true halotolerant enzyme, MmoC still showed 50 % of its specific activity at 2 M NaCl. We show that MmoC produces only trace amounts of superoxide, but mainly hydrogen peroxide during uncoupled turnover reactions. The characterization of a highly active reductase is an important step for future biotechnological applications of a halotolerant sMMO.     

Poly(vinylidene fluoride) ultrafiltration membranes containing hybrid silica nanoparticles: Preparation,characterization and performance

Poly(vinylidene fluoride) (PVDF) ultrafiltration membranes were prepared by immersion precipitation method using poly(hydroxyethyl methacrylate)-block-poly(methyl methacrylate) grafted silica (PHEMA-b-PMMA@SiO₂) nanoparticles as additives. The hybrid nanoparticles were synthesized by the surface initiated atom transfer radical polymerization (SI-ATRP), and they were characterized in detail by FT-IR, TEM, DLS and GPC. Results confirm that core–shell structure is formed after grafting PHEMA-b-PMMA brushes on the silica nanoparticles. Their average hydrodynamic diameter also increases with the prolongation of grafting time. After blending PVDF with the hybrid silica nanoparticles, the composite PVDF membranes exhibit high porosity and improved water permeation. Especially, when the molecular weight is 1.73 × 10⁵ g/mol for PHEMA-b-PMMA on the hybrid nanoparticles, the water flux of the PVDF composite membrane is 2.5 times than that of the control PVDF membrane, while the rejection to bovine serum albumin (BSA) remains at a high level (>90%). In addition, all the composite PVDF membranes show lower BSA adsorption and larger water flux recovery ratio than the control PVDF membrane. The improvement of membrane performance is attributed to the good hydrophilicity of PHEMA-b-PMMA@SiO₂ nanoparticles. Our results suggest that PHEMA-b-PMMA@SiO₂ nanoparticles with moderate molecular weight of PHEMA-b-PMMA are suitable for the property optimization of PVDF-based composite membranes.     

Interaction of platinum colloids with single crystalline oxide and graphite substrates: a combined AFM,STM and XPS study

The reactivity of ternary V₂O₅-WO₃/TiO₂ De-NO _x catalysts is investigated by transient and steady-state techniques, and results have been compared with those obtained over binary V₂O₅/TIO₂ samples having the same V₂O₅ loading. The results indicate that the reactivity of the ternary catalysts in the SCR reaction is higher than that of the vanadia-titania samples, and that at low temperatures the SCR reaction occurs via a redox mechanism that involves at first the participation of the catalyst lattice oxygen and then the reoxidation of the reduced sites by gas-phase oxygen. Accordingly, the higher reactivity of the ternary catalysts has been related to their superior redox properties.     

Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target

Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O₂ with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.     

Electrochemical study in both classical cell and microreactors of flavin adenine dinucleotide as a redox mediator for NADH regeneration

The electrochemical reduction of flavin adenine dinucleotide (FAD) is studied in a classical electrochemical cell as well as in two types of microreactors: the first one is a one-channel reactor and the other one, a multichannel filter-press reactor. The ultimate goal is to use the reduced form of flavin (FADH₂), in the presence of formate dehydrogenase (FDH), in order to continuously regenerate the reduced form of nicotinamide adenine dinucleotide (NADH) for chiral syntheses. Various voltammetric and adsorption measurements were carried out for a better understanding of the redox behavior of the FAD as well as its adsorption on gold. Diffusivity and kinetic electrochemical parameters of FAD were determined.     

Electron Beam Induced Reduction of V2O5 Studied by Analytical Electron Microscopy

The reduction of V₂O₅ under electron irradiation was studied by means of electron energy-loss spectroscopy, electron diffraction, and high-resolution imaging. The decrease of spectral intensity of O 1s excitations indicates a preferential removal of oxygen. The observed chemical shifts of the V 2p_3/2 and V 2p_1/2 peaks reveal that V⁵⁺ is reduced to V²⁺. Electron diffraction and high-resolution imaging show a structural change from the orthorhombic V₂O₅ to cubic VO. The beam induced reduction is compared with thermal decomposition of V₂O₅.     

Preparation of In,H-ZSM-5 for DeNOx reactions by solid-state ion exchange

A simple method is proposed to prepare In,H-ZSM-5 catalyst for DeNOx reactions. This consists of mechanically mixing the fine powders of In₂O₃ and H-ZSM-5 followed by heating in oxygen free inert gas flow to 580 °C where indium undergoes thermal auto-reduction and moves into exchange positions as In⁺ without destroying the crystalline structure of the zeolite.It was evidenced by IR, temperature-programmed reduction (TPR) and reoxidation that, once In⁺ was introduced into the lattice either by reductive solid-state ion exchange (RSSIE) or by thermal auto-reductive SSIE, it can be oxidized by O₂ or in the DeNOx reaction to (InO)⁺. The formed (InO)⁺ can easily be reduced to In⁺ suggesting that In,H-ZSM-5 might be a good catalyst for reactions where a redox cycle in the catalyst is involved in the reaction mechanism.Selective catalytic reduction (SCR) by methane proved that only a small fraction of In exchanged, together with some acid sites of the zeolite formed the active center for the catalytic reaction. XRD, XPS and FT-IR using pyridine proved that the structure of the zeolite and these centers are stable under reaction conditions and In is mainly in the form of (InO)⁺ in the used catalyst.