Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

The cytochrome b6f complex: plastoquinol oxidation and regulation of electron transport in chloroplasts.

In oxygenic photosynthetic systems, the cytochrome b6f (Cytb6f) complex (plastoquinol:plastocyanin oxidoreductase) is a heart of the hub that provides connectivity between photosystems (PS) II and I. In this review, the structure and function of the Cytb6f complex are briefly outlined, being focused on the mechanisms of a bifurcated (two-electron) oxidation of plastoquinol (PQH2). In plant chloroplasts, under a wide range of experimental conditions (pH and temperature), a diffusion of PQH2 from PSII to the Cytb6f does not limit the intersystem electron transport. The overall rate of PQH2 turnover is determined mainly by the first step of the bifurcated oxidation of PQH2 at the catalytic site Qo, i.e., the reaction of electron transfer from PQH2 to the Fe2S2 cluster of the high-potential Rieske iron-sulfur protein (ISP). This point has been supported by the quantum chemical analysis of PQH2 oxidation within the framework of a model system including the Fe2S2 cluster of the ISP and surrounding amino acids, the low-potential heme b6L, Glu78 and 2,3,5-trimethylbenzoquinol (the tail-less analog of PQH2). Other structure-function relationships and mechanisms of electron transport regulation of oxygenic photosynthesis associated with the Cytb6f complex are briefly outlined: pH-dependent control of the intersystem electron transport and the regulatory balance between the operation of linear and cyclic electron transfer chains.

Electron Transport in Chloroplasts: Regulation and Alternative Pathways of Electron Transfer.

This work represents an overview of electron transport regulation in chloroplasts as considered in the context of structure-function organization of photosynthetic apparatus in plants. Main focus of the article is on bifurcated oxidation of plastoquinol by the cytochrome b6f complex, which represents the rate-limiting step of electron transfer between photosystems II and I. Electron transport along the chains of non-cyclic, cyclic, and pseudocyclic electron flow, their relationships to generation of the trans-thylakoid difference in electrochemical potentials of protons in chloroplasts, and pH-dependent mechanisms of regulation of the cytochrome b6f complex are considered. Redox reactions with participation of molecular oxygen and ascorbate, alternative mediators of electron transport in chloroplasts, have also been discussed.

Mitochondrial Genome-Encoded Long Noncoding RNA Cytochrome B and Mitochondrial Dysfunction in Diabetic Retinopathy.

Aims: Mitochondrial dysfunction is closely associated with the development of diabetic complications. In diabetic retinopathy, electron transport chain is compromised and mitochondrial DNA (mtDNA) is damaged, downregulating transcription of mtDNA-encoded cytochrome B (CYTB) and its antisense long noncoding RNA, long noncoding RNA cytochrome B (LncCytB). Our goal was to investigate the role of LncCytB in the regulation of CYTB and mitochondrial function in diabetic retinopathy. Methods: Using human retinal endothelial cells, genetically manipulated for LncCytB (overexpression or silencing), the effect of high glucose (20 mM d-glucose) on LncCytB-CYTB interactions (by chromatin isolation by RNA purification), CYTB gene expression (by real-time quantitative polymerase chain reaction), complex III activity, mitochondrial free radicals, and oxygen consumption rate (OCR, by Seahorse XF analyzer) was investigated. Key results were confirmed in the retinal microvessels from streptozotocin-induced diabetic mice. Results: High glucose decreased LncCytB-CYTB interactions, and while LncCytB overexpression ameliorated glucose-induced decrease in CYTB gene transcripts, complex III activity and OCR and increase in mitochondrial reactive oxygen species, LncCytB-siRNA further attenuated CYTB gene transcription, complex III activity, and OCR. Similar decrease in LncCytB-CYTB interactions and CYTB transcription was observed in diabetic mice. Furthermore, maintenance of mitochondrial homeostasis by overexpressing superoxide dismutase or sirtuin 1 in mice ameliorated diabetes-induced decrease in LncCytB-CYTB interactions and CYTB gene transcripts, and also improved complex III activity and mitochondrial respiration. Innovation and Conclusion: LncCytB downregulation in hyperglycemic milieu downregulates CYTB transcription, which inhibits complex III activity and compromises mitochondrial stability and OCR. Thus, preventing LncCytB downregulation in diabetes has potential of inhibiting the development of diabetic retinopathy, possibly via maintaining mitochondrial respiration. Antioxid. Redox Signal. 39, 817-828.

Far-red photosynthesis: Two charge separation pathways exist in plant Photosystem II reaction center.

An alternative charge separation pathway in Photosystem II under the far-red light was proposed by us on the basis of electron transfer properties at 295 K and 5 K. Here we extend these studies to the temperature range of 77-295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of Cytochrome b559 and ChlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730-750 nm) spectral regions. Temperature dependence of the S2 state signal induction after single flash at 730-750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data indicate that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in Photosystem II with localization of hole on the ChlD1 molecule.

Role of the phospholipid binding sites, PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2.

In phagocytes, superoxide anion (O2-), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme. To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1-286), p67phox(aa 1-212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.

Increased metabolism in combination with the novel cytochrome b target-site mutation L258F confers cross-resistance between the Qo inhibitors acequinocyl and bifenazate in Tetranychus urticae.

Acequinocyl and bifenazate are potent acaricides acting at the Qo site of complex III of the electron transport chain, but frequent applications of these acaricides have led to the development of resistance in spider mites. Target-site resistance caused by mutations in the conserved cd1- and ef-helices of the Qo pocket of cytochrome b has been elucidated as the main resistance mechanism. We therefore monitored Qo pocket mutations in European field populations of Tetranychus urticae and uncovered a new mutation, L258F. The role of this mutation was validated by revealing patterns of maternal inheritance and by the independently replicated introgression in an unrelated susceptible genetic background. However, the parental strain exhibited higher resistance levels than conferred by the mutation alone in isogenic lines, especially for acequinocyl, implying the involvement of strong additional resistance mechanisms. This was confirmed by revealing a polygenic inheritance pattern with classical genetic crosses and via synergism experiments. Therefore, a genome-wide expression analysis was conducted that identified a number of highly overexpressed detoxification genes, including many P450s. Functional expression revealed that the P450 CYP392A11 can metabolize bifenazate by hydroxylation of the ring structure. In conclusion, the novel cytochrome b target-site mutation L258F was uncovered in a recently collected field strain and its role in acequinocyl and bifenazate resistance was validated. However, the high level of resistance in this strain is most likely caused by a combination of target-site resistance and P450-based increased detoxification, potentially acting in synergism.

The cytochrome b carboxyl terminal region is necessary for mitochondrial complex III assembly.

Mitochondrial bc 1 complex from yeast has 10 subunits, but only cytochrome b (Cytb) subunit is encoded in the mitochondrial genome. Cytb has eight transmembrane helices containing two hemes b for electron transfer. Cbp3 and Cbp6 assist Cytb synthesis, and together with Cbp4 induce Cytb hemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cytb synthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Because Qcr7 resides near the Cytb carboxyl region, we wondered whether this region is important for Cytb synthesis/assembly. Although deletion of the Cytb C-region did not abrogate Cytb synthesis, the assembly-feedback regulation was lost, so Cytb synthesis was normal even if Qcr7 was missing. Mutants lacking the Cytb C-terminus were non-respiratory because of the absence of fully assembled bc 1 complex. By performing complexome profiling, we showed the existence of aberrant early-stage subassemblies in the mutant. In this work, we demonstrate that the C-terminal region of Cytb is critical for regulation of Cytb synthesis and bc 1 complex assembly.

[Geometric Approach to Phylogeographic Analysis Molecular Genetic Sequences: Principal Components and Dendrograms].

Currently, the search for manifestations of selection under the influence of the environment in molecular sequences is usually carried out within closely related species or at the intraspecific level. It is believed that at high taxonomic levels this is unpromising due to phylogenetic relationship. Cytochrome b amino acid sequences of 67 rodent and lagomorph species with known geographic coordinates were digitized using the AAindex database. Based on more than 200 thousand characters, the principal components were obtained. A well-known statistical method, which has not been previously used for such problems, was used, which makes it possible to orthogonally decompose multidimensional variability into intra- and intertaxon variability and analyze them separately. The subfamily level was selected. For the second principal component (17.05% of intertaxon variability), a correlation with latitude was found (r = 0.561; n = 67; p < E-5). The clear division into two groups, revealed by the first principal component (39.48% of intertaxon variability), which does not coincide with the taxonomic one, indicates a possible physicochemical underlying cause for the differences between them. This requires further research.

Exploration of Novel Scaffolds Targeting Cytochrome b of Pyricularia oryzae.

The fulfilment of the European "Farm to Fork" strategy requires a drastic reduction in the use of "at risk" synthetic pesticides; this exposes vulnerable agricultural sectors-among which is the European risiculture-to the lack of efficient means for the management of devastating diseases, thus endangering food security. Therefore, novel scaffolds need to be identified for the synthesis of new and more environmentally friendly fungicides. In the present work, we employed our previously developed 3D model of P. oryzae cytochrome bc1 (cyt bc1) complex to perform a high-throughput virtual screening of two commercially available compound libraries. Three chemotypes were selected, from which a small collection of differently substituted analogues was designed and synthesized. The compounds were tested as inhibitors of the cyt bc1 enzyme function and the mycelium growth of both strobilurin-sensitive (WT) and -resistant (RES) P. oryzae strains. This pipeline has permitted the identification of thirteen compounds active against the RES cyt bc1 and five compounds that inhibited the WT cyt bc1 function while inhibiting the fungal mycelia only minimally. Serendipitously, among the studied compounds we identified a new chemotype that is able to efficiently inhibit the mycelium growth of WT and RES strains by ca. 60%, without inhibiting the cyt bc1 enzymatic function, suggesting a different mechanism of action.

The evolution of the human mitochondrial bc1 complex- adaptation for reduced rate of superoxide production?

The mitochondrial bc1 complex is a major source of mitochondrial superoxide. While bc1-generated superoxide plays a beneficial signaling role, excess production of superoxide lead to aging and degenerative diseases. The catalytic core of bc1 comprises three peptides -cytochrome b, Fe-S protein, and cytochrome c1. All three core peptides exhibit accelerated evolution in anthropoid primates. It has been suggested that the evolution of cytochrome b in anthropoids was driven by a pressure to reduce the production of superoxide. In humans, the bc1 core peptides exhibit anthropoid-specific substitutions that are clustered near functionally critical sites that may affect the production of superoxide. Here we compare the high-resolution structures of bovine, mouse, sheep and human bc1 to identify structural changes that are associated with human-specific substitutions. Several cytochrome b substitutions in humans alter its interactions with other subunits. Most significantly, there is a cluster of seven substitutions, in cytochrome b, the Fe-S protein, and cytochrome c1 that affect the interactions between these proteins at the tether arm of the Fe-S protein and may alter the rate of ubiquinone oxidation and the rate of superoxide production. Another cluster of substitutions near heme bH and the ubiquinone reduction site, Qi, may affect the rate of ubiquinone reduction and thus alter the rate of superoxide production. These results are compatible with the hypothesis that cytochrome b in humans (and other anthropoid primates) evolve to reduce the rate of production of superoxide thus enabling the exceptional longevity and exceptional cognitive ability of humans.

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1).

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.

High-resolution cryo-EM structures of plant cytochrome b6f at work.

Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b6f (Cyt b6f) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Qp) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b6f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Qp in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b6f in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.

Photoinduced electron transfer in cytochrome bc1: Dynamics of rotation of the Iron-sulfur protein during bifurcated electron transfer from ubiquinol to cytochrome c1 and cytochrome bL.

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc1 (cyt bc1) were studied using a binuclear ruthenium complex to rapidly photooxidize cyt c1. When cyt c1, the iron­sulfur center Fe2S2, and cyt bH were reduced before the reaction, photooxidation of cyt c1 led to electron transfer from Fe2S2 to cyt c1 with a rate constant of ka = 80,000 s-1, followed by bifurcated reduction of both Fe2S2 and cyt bL by QH2 in the Qo site with a rate constant of k2 = 3000 s-1. The resulting Q then traveled from the Qo site to the Qi site and oxidized one equivalent each of cyt bL and cyt bH with a rate constant of k3 = 340 s-1. The rate constant ka was decreased in a nonlinear fashion by a factor of 53 as the viscosity was increased to 13.7. A mechanism that is consistent with the effect of viscosity involves rotational diffusion of the iron­sulfur protein from the b state with reduced Fe2S2 close to cyt bL to one or more intermediate states, followed by rotation to the final c1 state with Fe2S2 close to cyt c1, and rapid electron transfer to cyt c1.

Quantification of energy-converting protein complexes in plant thylakoid membranes.

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b6f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.

The thylakoid membrane protein NTA1 is an assembly factor of the cytochrome b6f complex essential for chloroplast development in Arabidopsis.

The cytochrome b6f (Cyt b6f) complex is a multisubunit protein complex in chloroplast thylakoid membranes required for photosynthetic electron transport. Here we report the isolation and characterization of the new tiny albino 1 (nta1) mutant in Arabidopsis, which has severe defects in Cyt b6f accumulation and chloroplast development. Gene cloning revealed that the nta1 phenotype was caused by disruption of a single nuclear gene, NTA1, which encodes an integral thylakoid membrane protein conserved across green algae and plants. Overexpression of NTA1 completely rescued the nta1 phenotype, and knockout of NTA1 in wild-type plants recapitulated the mutant phenotype. Loss of NTA1 function severely impaired the accumulation of multiprotein complexes related to photosynthesis in thylakoid membranes, particularly the components of Cyt b6f. NTA1 was shown to directly interact with four subunits (Cyt b6/PetB, PetD, PetG, and PetN) of Cyt b6f through the DUF1279 domain and C-terminal sequence to mediate their assembly. Taken together, our results identify NTA1 as a new and key regulator of chloroplast development that plays essential roles in assembly of the Cyt b6f complex by interacting with multiple Cyt b6f subunits.

Rieske FeS overexpression in tobacco provides increased abundance and activity of cytochrome b6 f.

Photosynthesis is fundamental for plant growth and yield. The cytochrome b6 f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of cytochrome b6 f, the Rieske FeS protein, led to up to a 40% increase in the abundance of the complex in Nicotiana tabacum (tobacco) and was accompanied by an enhanced in vitro cytochrome f activity, indicating a full functionality of the complex. Analysis of transgenic plants overexpressing Rieske FeS by the light-induced fluorescence transients technique revealed a more oxidised primary quinone acceptor of photosystem II (QA ) and plastoquinone pool and faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance, compared to control plants. A faster establishment of qE , the energy-dependent component of nonphotochemical quenching, in transgenic plants suggests a more rapid buildup of the transmembrane proton gradient, also supporting the increased in vivo cytochrome b6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO2 assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances the abundance of functional cytochrome b6 f and may have the potential to increase plant productivity if combined with other traits.

Biocatalytic system for comparatively assessing the functional association of monolignol cytochrome P450 monooxygenases with their redox partners.

Lignin is a complex heterogenous polymer derived from oxidative radical polymerization of three monolignols, i.e., p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These lignin monomeric precursors structurally differ in their methoxy groups of the benzene rings. In phenylpropanoid-monolignol biosynthetic pathway, the endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, cinnamate 4-hydroxylase, coumaroyl ester 3'-hydroxylase and ferulate 5-hydroxylase, establish the key structural characteristics of monolignols. The catalysis of cytochrome P450 monooxygenase requires reducing power, which is supplied by the ER electron transfer chains, composed of cytochrome P450 oxidoreductase (CPR), cytochrome b5 reductase (CBR) and/or cytochrome b5 protein (CB5), from cofactor NADPH or NADH. While NADPH-dependent CPR serves as the typical electron donor for most P450 enzymes, in some cases, the CBR-CB5 or CPR-CB5 electron transfer system also transfers electrons to the terminal P450 enzymes. There are tremendous studies focusing on the discovery and characterization of cytochrome P450 monooxygenases. However, very limited attention has been paid to the versatility and the roles of electron transfer components in the P450 catalytic system. Due to the membrane-residence property of both P450 enzymes and electron transfer components, it is challenging to establish an effective experimental system to evaluate the functional association of P450s with their redox partners. This chapter describes a yeast cell biocatalytic system and the related experimental procedures for comparatively assessing the functional relationship of monolignol biosynthetic P450 enzymes and different redox partners in their catalysis.

Plastoquinol Oxidation: Rate-Limiting Stage in the Electron Transport Chain of Chloroplasts.

This work is devoted to theoretical study of functioning of the cytochrome (Cyt) b6f complex (plastoquinol:plastocyanin oxidoreductase) of the electron transport chain (ETC) in oxygenic photosynthesis. A composition of the chloroplast ETC and molecular mechanisms of functioning of the Cyt b6f complex, which stands between photosystems II and I (PSII and PSI), are briefly reviewed. The Cyt b6f complex oxidizes plastoquinol (PQH2) molecules formed in PSII, and reduces plastocyanin, which serves as an electron donor to PSI. PQH2 oxidation is the rate-limiting step in the chain of electron transfer processes between PSII and PSI. Using the density functional theory (DFT) method, we have analyzed the two-electron (bifurcated) oxidation of PQH2 in the catalytic center Qo of the Cyt b6f complex. Results of DFT calculations are consistent with the fact that the first step of PQH2 oxidation, electron transfer to the Fe2S2 cluster of the iron-sulfur protein (ISP), is an endergonic (energy-accepting) process (ΔE ≈ 15 kJ·mol-1) that can limit turnover of the Cyt b6f complex. The second stage of bifurcated oxidation of PQH2 - electron transfer from semiquinone (PQH•, formed after the first step of PQH2 oxidation) to heme b6L - is the exergonic (energy-donating) process (ΔE < 0). DFT modeling of this stage revealed that semiquinone oxidation should accelerate after the PQH• radical shift towards the heme b6L (an electron acceptor) and the carboxy group of Glu78 (a proton acceptor). The data obtained are discussed within the framework of the Mitchell Q-cycle model describing PQH2 oxidation at the Qo site of the Cyt b6f complex.

The G143A/S substitution of mitochondrially encoded cytochrome b (Cytb) in Magnaporthe oryzae confers resistance to quinone outside inhibitors.

BACKGROUND: Rice blast, caused by Magnaporthe oryzae, is a destructive disease threatening the production of staple foods worldwide. Quinone outside inhibitors (QoIs) are a group of chemicals exhibiting excellent activity against a majority of plant pathogens, with the disadvantage that pathogens can easily develop resistance to QoIs. RESULTS: Here, we investigated the activity of picoxystrobin against M. oryzae, which showed a great inhibitory effect on 100 strains of M. oryzae with half-maximal effective concentrations (EC50 ) ranging from 0.0251 to 0.1337 µg ml-1 . The EC50 values showed a continuous unimodal distribution that was identical to the normal distribution, suggesting the potency of our study to represent baseline sensitivity. In addition, nine resistant mutants were obtained by exposing M. oryzae to a high dosage of picoxystrobin in the laboratory; all of them showed cross-resistance to the other five QoI fungicides. Although some mutants showed a decreased resistance factor after ten successive cultures on fungicide-free medium, the resistance to picoxystrobin was still inheritable. Amino acid substitution of G143S was detected in eight of nine picoxystrobin-resistant mutants, and G143A was detected in only one of nine mutants. A fitness penalty was found in the mutants carrying G143S rather than G143A. CONCLUSION: Our findings suggested that M. oryzae had a mid to high risk of resistance to picoxystrobin. Considering this, we should be vigilant to the resistance risk and apply picoxystrobin sensibly in the field. © 2022 Society of Chemical Industry.