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Malaria remains a global health concern, with the emergence of resistance to the antimalarial drug atovaquone through cytochrome b (cyt b) being well-documented. This study was prompted by the presence of this mutation in cyt b to enable new drug candidates capable of overcoming drug resistance. Our objective was to identify potential drug candidates from compounds of Xylocarpus granatum by computationally assessing their interactions with Plasmodium berghei cyt b. Using computational methods, we modeled cyt b (GenBank: AF146076.1), identified the binding cavity, and analyzed the Ramachandran plot against cyt b. Additionally, we conducted drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies, along with density functional theory (DFT) analysis of the compounds. Molecular docking and molecular dynamics simulation (MDS) were used to evaluate the binding energy and stability of the cyt b-ligand complex. Notably, our investigation highlighted kaempferol as a promising compound due to its high binding energy of 7.67 kcal/mol among all X. granatum compounds, coupled with favorable pharmacological properties (ADMET) and antiprotozoal properties at Pa 0.345 > Pi 0.009 (PASS value). DFT analysis showed that kaempferol has an energy gap of 4.514 eV. MDS indicated that all tested ligands caused changes in bonding and affected the structural conformation of cyt b, as observed before MDS (0 ns) and after MDS (100 ns). The most notable differences were observed in the types of hydrogen bonds between 0 and 100 ns. Nevertheles, MDS results from a 100 ns simulation revealed consistent behavior for kaempferol across various parameters including root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), molecular mechanics-Poisson Boltzmann surface area (MM-PBSA), and hydrogen bonds. The cyt b-kaempferol complex demonstrated favorable energy stability, as supported by the internal energy distribution values observed in principal component analysis (PCA), which closely resembled those of the atovaquone control. Additionally, trajectory stability analysis indicated structural stability, with a cumulative eigenvalue of 24.7 %. Dynamic cross-correlation matrix (DCCM) analysis revealed a positive correlation among catalytic cytochrome residues within the amino acid residues range 119-268. The results of our research indicate that the structure of kaempferol holds promise as a potential candidate against Plasmodium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The natural product 9-methoxystrobilurin G (9MG) from Favolaschia spp basidiomycetes is a potent and selective antimalarial. The mechanism of action of 9MG is unknown. We induced 9MG resistance in Plasmodium falciparum 3D7 and Dd2 strains and identified mutations associated with resistance by genome sequencing. All 9MG-resistant clones possessed missense mutations in the cytochrome b (CYTB) gene, a key component of mitochondrial complex III. The mutations map to the quinol oxidation site of CYTB, which is also the target of antimalarials such as atovaquone. In a complementary approach to identify protein targets of 9MG, a photoactivatable derivative of 9MG was synthesized and applied in chemoproteomic-based target profiling. Three components of mitochondrial complex III (QCR7, QCR9, and COX15) were specifically enriched consistent with 9MG targeting CYTB and complex III function in P. falciparum . Inhibition of complex III activity by 9MG was confirmed by ubiquinone cytochrome c reductase assay using P. falciparum extract. The findings from this study may be useful for developing novel antimalarials targeting CYTB.

Background: Wheat crown rot (WCR), primarily caused by Fusarium pseudograminearum has become more and more prevalent in winter wheat areas in China. However, limited fungicides have been registered for the control of WCR in China so far. Pyraclostrobin is a representative quinone outside inhibitor (QoI) with excellent activity against Fusarium spp. There is currently limited research on the resistance risk and resistance mechanism of F. pseudograminearum to pyraclostrobin., Results: Here, we determined the activity of pyraclostrobin against F. pseudograminearum. The EC 50 values ranged from 0.022 to 0.172 μg mL -1 with an average EC 50 value of 0.071 ± 0.030 μg mL -1 . Four highly pyraclostrobin-resistant mutants were obtained from two sensitive strains by ultraviolet (UV) mutagenesis in the laboratory. The mutants showed decreased mycelial growth rate and virulence as compared with the corresponding wild-type strains, indicating that pyraclostrobin resistance suffered a fitness penalty in F. pseudograminearum. It was found that the high resistance of four mutants was caused by the G143S mutation in Cytb. Molecular docking analysis also further confirms that the G143S mutation in Cytb decreased the binding affinity between pyraclostrobin and Cytb., Conclusion: The resistance risk of F. pseudograminearum to pyraclostrobin could be low to medium. Although a mutation at the G143S position of Cytb could potentially occur, this mutation decreases the fitness of the mutant, which may reduce its survival in the environment. Therefore, the negative consequences of a possible mutation are lower. This makes pyraclostrobin a good candidate for controlling crown rot in wheat. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Bifenazate, a potent acaricide that targets mitochondrial complex III, exhibits selective toxicity (>280-fold) toward phytophagous mites versus predatory mites. Here, a systematic study was conducted to clarify the selective mechanism. Nontarget factors were excluded through epidermal penetration tests and assessment of detoxification enzymes' activities. Quantification of IC 50 values, ATP content, and reactive oxygen species (ROS) levels revealed that differences in drug-target binding determine the toxicity selectivity. Structural modeling and molecular docking revealed that variations in key amino acid sites within the cytochrome b (cytb) target might regulate this selectivity, which was validated through a microscale thermophoresis assay. Significant disparities were observed in the binding affinity between bifenazate and recombinant cytb proteins derived from phytophagous mites and predatory mites. Mutating isoleucine 139 to leucine notably reduced the binding affinity of bifenazate to cytb. Insights into bifenazate selectivity between phytophagous and predatory mites inform a basis for developing compounds that target cytochrome b .

In diabetic retinopathy, mitochondrial DNA (mtDNA) is damaged and mtDNA-encoded genes and long noncoding RNA cytochrome B (LncCytB) are downregulated. LncRNAs lack an open reading frame, but they can regulate gene expression by associating with DNA/RNA/protein. Double stranded mtDNA has promoters on both heavy (HSP) and light (LSP) strands with binding sites for mitochondrial transcription factor A (TFAM) between them. The aim was to investigate the role of LncCytB in mtDNA transcription in diabetic retinopathy. Using human retinal endothelial cells incubated in high glucose, the effect of regulation of LncCytB on TFAM binding at mtDNA promoters was investigated by Chromatin immunoprecipitation, and binding of LncCytB at TFAM by RNA immunoprecipitation and RNA fluorescence in situ hybridization. High glucose decreased TFAM binding at both HSP and LSP, and binding of LncCytB at TFAM. While LncCytB overexpression ameliorated decrease in TFAM binding and transcription of genes encoded by both H- and L- strands, LncCytB-siRNA further downregulated them. Maintenance of mitochondrial homeostasis by overexpressing mitochondrial superoxide dismutase or Sirtuin-1 protected diabetes-induced decrease in TFAM binding at mtDNA and LncCytB binding at TFAM, and mtDNA transcription. Similar results were obtained from mouse retinal microvessels from streptozotocin-induced diabetic mice. Thus, LncCytB facilitates recruitment of TFAM at HSP and LSP, and its downregulation in diabetes compromises the binding, resulting in the downregulation of polypeptides encoded by mtDNA. Regulation of LncCytB, in addition to protecting mitochondrial genomic stability, should also help in maintaining the transcription of mtDNA encoded genes and electron transport chain integrity in diabetic retinopathy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The mitochondrial genome transcribes 13 mRNAs coding for well-known proteins essential for oxidative phosphorylation. We demonstrate here that cytochrome b (CYTB), the only mitochondrial-DNA-encoded transcript among complex III, also encodes an unrecognized 187-amino-acid-long protein, CYTB-187AA, using the standard genetic code of cytosolic ribosomes rather than the mitochondrial genetic code. After validating the existence of this mtDNA-encoded protein arising from cytosolic translation (mPACT) using mass spectrometry and antibodies, we show that CYTB-187AA is mainly localized in the mitochondrial matrix and promotes the pluripotent state in primed-to-naive transition by interacting with solute carrier family 25 member 3 (SLC25A3) to modulate ATP production. We further generated a transgenic knockin mouse model of CYTB-187AA silencing and found that reduction of CYTB-187AA impairs females' fertility by decreasing the number of ovarian follicles. For the first time, we uncovered the novel mPACT pattern of a mitochondrial mRNA and demonstrated the physiological function of this 14 th protein encoded by mtDNA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Membrane proteins carrying redox cofactors are key subunits of respiratory chain complexes, yet the exact path of their folding and maturation remains poorly understood. Here, using cryo-EM and structure prediction via Alphafold2, we generated models of early assembly intermediates of cytochrome b (Cytb), a central subunit of complex III. The predicted structure of the first assembly intermediate suggests how the binding of Cytb to the assembly factor Cbp3-Cbp6 imposes an open configuration to facilitate the acquisition of its heme cofactors. Moreover, structure predictions of the second intermediate indicate how hemes get stabilized by binding of the assembly factor Cbp4, with a concomitant weakening of the contact between Cbp3-Cbp6 and Cytb, preparing for the release of the fully hemylated protein from the assembly factors., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In oxygenic photosynthetic systems, the cytochrome b 6 f (Cytb 6 f) 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 Cytb 6 f complex are briefly outlined, being focused on the mechanisms of a bifurcated (two-electron) oxidation of plastoquinol (PQH 2 ). In plant chloroplasts, under a wide range of experimental conditions (pH and temperature), a diffusion of PQH 2 from PSII to the Cytb 6 f does not limit the intersystem electron transport. The overall rate of PQH 2 turnover is determined mainly by the first step of the bifurcated oxidation of PQH 2 at the catalytic site Q o , i.e., the reaction of electron transfer from PQH 2 to the Fe 2 S 2 cluster of the high-potential Rieske iron-sulfur protein (ISP). This point has been supported by the quantum chemical analysis of PQH 2 oxidation within the framework of a model system including the Fe 2 S 2 cluster of the ISP and surrounding amino acids, the low-potential heme b 6 L , Glu78 and 2,3,5-trimethylbenzoquinol (the tail-less analog of PQH 2 ). Other structure-function relationships and mechanisms of electron transport regulation of oxygenic photosynthesis associated with the Cytb 6 f 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., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: The authors declare no conflict of interest.

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 S 2 state multiline signal, oxidation of Cytochrome b 559 and Chlorophyll Z 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 S 2 state signal induction after single flash at 730-750 nm (T inhibition  ~ 240 K) was found to be different than that at 532 nm (T inhibition  ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b 559 or chlorophyll Z 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 Y Z /CaMn 4 O 5 -cluster oxidation):(yield of Cytb 559 /Chl Z /Car D2 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 Chl D1 molecule., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stenbjorn Styring reports financial support was provided by Swedish Energy Agency., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 (Lnc CytB ). Our goal was to investigate the role of Lnc CytB in the regulation of CYTB and mitochondrial function in diabetic retinopathy. Methods: Using human retinal endothelial cells, genetically manipulated for Lnc CytB (overexpression or silencing), the effect of high glucose (20 m M d-glucose) on Lnc CytB - 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 Lnc CytB-CYTB interactions, and while Lnc CytB overexpression ameliorated glucose-induced decrease in CYTB gene transcripts, complex III activity and OCR and increase in mitochondrial reactive oxygen species, Lnc CytB-siRNA further attenuated CYTB gene transcription, complex III activity, and OCR. Similar decrease in Lnc CytB - 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 Lnc CytB - CYTB interactions and CYTB gene transcripts, and also improved complex III activity and mitochondrial respiration. Innovation and Conclusion: Lnc CytB downregulation in hyperglycemic milieu downregulates CYTB transcription, which inhibits complex III activity and compromises mitochondrial stability and OCR. Thus, preventing Lnc CytB downregulation in diabetes has potential of inhibiting the development of diabetic retinopathy, possibly via maintaining mitochondrial respiration. Antioxid. Redox Signal. 39, 817-828.

In phagocytes, superoxide anion (O 2 - ), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b 558 (cyt b 558 ) and four cytosolic components: p40 phox , p47 phox , p67 phox , 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 b 558 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 p47 phox . We also used a fused chimera consisting of p47 phox (aa 1-286), p67 phox (aa 1-212) and Rac1Q61L, as well as mutated versions in the p47 phox 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 b 558 . They also have an impact on O 2 .- 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 b 6 f 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 b 6 f complex are considered. Redox reactions with participation of molecular oxygen and ascorbate, alternative mediators of electron transport in chloroplasts, have also been discussed.

Acequinocyl and bifenazate are potent acaricides acting at the Q o 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 Q o pocket of cytochrome b has been elucidated as the main resistance mechanism. We therefore monitored Q o 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mitochondrial bc 1 complex from yeast has 10 subunits, but only cytochrome b (Cyt b ) subunit is encoded in the mitochondrial genome. Cyt b has eight transmembrane helices containing two hemes b for electron transfer. Cbp3 and Cbp6 assist Cyt b synthesis, and together with Cbp4 induce Cyt b hemylation. Subunits Qcr7/Qcr8 participate in the first steps of assembly, and lack of Qcr7 reduces Cyt b synthesis through an assembly-feedback mechanism involving Cbp3/Cbp6. Because Qcr7 resides near the Cyt b carboxyl region, we wondered whether this region is important for Cyt b synthesis/assembly. Although deletion of the Cyt b C-region did not abrogate Cyt b synthesis, the assembly-feedback regulation was lost, so Cyt b synthesis was normal even if Qcr7 was missing. Mutants lacking the Cyt b 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 Cyt b is critical for regulation of Cyt b synthesis and bc 1 complex assembly., (© 2023 Flores-Mireles et al.)

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 b 6 f complex (cyt b 6 f 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 b 6 f 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., Competing Interests: Declaration of competing interest Helmut Kirchhoff reports financial support was provided by US Department of Energy and the National Science Foundation USA., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc 1 (cyt bc 1 ) were studied using a binuclear ruthenium complex to rapidly photooxidize cyt c 1 . When cyt c 1 , the iron‑sulfur center Fe 2 S 2 , and cyt b H were reduced before the reaction, photooxidation of cyt c 1 led to electron transfer from Fe 2 S 2 to cyt c 1 with a rate constant of k a  = 80,000 s -1 , followed by bifurcated reduction of both Fe 2 S 2 and cyt b L by QH 2 in the Q o site with a rate constant of k 2  = 3000 s -1 . The resulting Q then traveled from the Q o site to the Q i site and oxidized one equivalent each of cyt b L and cyt b H with a rate constant of k 3  = 340 s -1 . The rate constant k a 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 iron‑sulfur protein from the b state with reduced Fe 2 S 2 close to cyt b L to one or more intermediate states, followed by rotation to the final c 1 state with Fe 2 S 2 close to cyt c 1 , and rapid electron transfer to cyt c 1 ., Competing Interests: Declaration of competing interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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 Ni 2+ -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 b 5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (k cat /K m ) 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 b 5 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 b 5 protein in the lyase reactions., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b 6 f (Cyt b 6 f) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Q p ) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b 6 f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Q p in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b 6 f 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.

The cytochrome b 6f  (Cyt b 6f ) 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 b 6f  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 b 6f . NTA1 was shown to directly interact with four subunits (Cyt b 6 /PetB, PetD, PetG, and PetN) of Cyt b 6f  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 b 6f  complex by interacting with multiple Cyt b 6f  subunits., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Life-long efforts of the Tartu photosynthesis research group have been summarized. The measurements were facilitated by self-designed instruments, distinct in multifunctionality and fastresponse time. The black-box type kinetical analysis on intact leaves has revealed several physiologically significant features of leaf photosynthesis. Rubisco studies reflected competition for the active site between the substrates and products, linearizing in vivo kinetics compared with the low-K m in vitro responses. Rubisco Activase usually activates only a small part of the Rubisco, making the rest of it a storage protein. Precisely quantifying absorbed photons and the responding transmittance changes, electron flow rates through cytochrome b 6 f, plastocyanin and photosystem I were measured, revealing competition between the proton-uncoupled cyclic electron flow from PSI to Cyt b 6 f to P700 + and the proton-coupled linear flow from PSII to Cyt b 6 f to P700 + . Analyzing responses of O 2 evolution and Chl fluorescence to ms-length light pulses we concluded that explanation of the sigmoidal fluorescence induction by excitonic connectivity between PSII units is a misconception. Each PSII processes excitation from its own antenna, but the sigmoidicity is caused by rise of the fluorescence yield of the Q A -reduced PSII units after their Q B site becomes occupied by reduced plastoquinone (or diuron). Unlike respiration, photosynthetic electrons must prepare their acceptor by coupled synthesis of 3ATP/4e - . Feedback regulation of this ratio leads to oscillations under saturating light and CO 2 , when the rate is P i -limited. The slow oscillations (period 60s) indicate that the magnitudes of the deflections in the 3ATP/4e - ratio, corrected by regulating cyclic and alternative electron flow (including the Mehler type O 2 reduction), are only a fraction of a per cent. The P i limitation causes slip in the ATP synthase, slightly increasing the basic 12H + /3ATP requirement., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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 (EC 50 ) ranging from 0.0251 to 0.1337 μg ml -1 . The EC 50 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., (© 2022 Society of Chemical Industry.)

Photosynthesis is fundamental for plant growth and yield. The cytochrome b 6 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 b 6 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 (Q A ) 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 q E , 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 b 6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO 2 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 b 6 f and may have the potential to increase plant productivity if combined with other traits., (© 2022 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Methane produced by methanogenic archaea has an important influence on Earth's changing climate. Methanogenic archaea are phylogenetically diverse and widespread in anoxic environments. These microorganisms can be divided into two subgroups based on whether or not they use b-type cytochromes for energy conservation. Methanogens with b-type cytochromes have a wider substrate range and higher growth yields than those without them. To date, methanogens with b-type cytochromes were found exclusively in the phylum "Ca. Halobacteriota" (formerly part of the phylum Euryarchaeota). Here, we present the discovery of metagenome-assembled genomes harboring methyl-coenzyme M reductase genes reconstructed from mesophilic anoxic sediments, together with the previously reported thermophilic "Ca. Methylarchaeum tengchongensis", representing a novel archaeal order, designated the "Ca. Methylarchaeales", of the phylum Thermoproteota (formerly the TACK superphylum). These microorganisms contain genes required for methyl-reducing methanogenesis and the Wood-Ljundahl pathway. Importantly, the genus "Ca. Methanotowutia" of the "Ca. Methylarchaeales" encode a cytochrome b-containing heterodisulfide reductase (HdrDE) and methanophenazine-reducing hydrogenase complex that have similar gene arrangements to those found in methanogenic Methanosarcinales. Our results indicate that members of the "Ca. Methylarchaeales" are methanogens with cytochromes and can conserve energy via membrane-bound electron transport chains. Phylogenetic and amalgamated likelihood estimation analyses indicate that methanogens with cytochrome b-containing electron transfer complexes likely evolved before diversification of Thermoproteota or "Ca. Halobacteriota" in the early Archean Eon. Surveys of public sequence databases suggest that members of the lineage are globally distributed in anoxic sediments and may be important players in the methane cycle., (© 2022. The Author(s).)

Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy. Abbreviations: Apob : apolipoprotein B; Atg1 : autophagy-related 1; Atg7 : autophagy related 7; Atp5a1 : ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3 : BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8 : mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3 : mitochondrially encoded cytochrome c oxidase III; mt-Cytb : mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9 : NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt : nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2 : polymerase (DNA directed), gamma 2, accessory subunit; Ppara : peroxisome proliferator activated receptor alpha; Ppia : peptidylprolyl isomerase A; Prkn : parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19 : ribosomal protein L19; Rps18 : ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1 : single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m : transcription factor B1, mitochondrial; Tfb2m : transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.

The acclimation of higher plants to different light intensities is associated with a reorganization of the photosynthetic apparatus. These modifications, namely, changes in the amount of peripheral antenna (LHCII) of photosystem (PS) II and changes in PSII/PSI stoichiometry, typically lead to an altered chlorophyll (Chl) a/b ratio. However, our previous studies show that in spruce, this ratio is not affected by changes in growth light intensity. The evolutionary loss of PSII antenna proteins LHCB3 and LHCB6 in the Pinaceae family is another indication that the light acclimation strategy in spruce could be different. Here we show that, unlike Arabidopsis, spruce does not modify its PSII/PSI ratio and PSII antenna size to maximize its photosynthetic performance during light acclimation. Its large PSII antenna consists of many weakly bound LHCIIs, which form effective quenching centers, even at relatively low light. This, together with sensitive photosynthetic control on the level of cytochrome b 6 f complex (protecting PSI), is the crucial photoprotective mechanism in spruce. High-light acclimation of spruce involves the disruption of PSII macro-organization, reduction of the amount of both PSII and PSI core complexes, synthesis of stress proteins that bind released Chls, and formation of "locked-in" quenching centers from uncoupled LHCIIs. Such response has been previously observed in the evergreen angiosperm Monstera deliciosa exposed to high light. We suggest that, in contrast to annuals, shade-tolerant evergreen land plants have their own strategy to cope with light intensity changes and the hallmark of this strategy is a stable Chl a/b ratio., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

This work is devoted to theoretical study of functioning of the cytochrome (Cyt) b 6 f 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 b 6 f complex, which stands between photosystems II and I (PSII and PSI), are briefly reviewed. The Cyt b 6 f 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 Q o of the Cyt b 6 f 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 b 6 f complex. The second stage of bifurcated oxidation of PQH2 - electron transfer from semiquinone (PQH • , formed after the first step of PQH2 oxidation) to heme b 6 L - 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 b 6 L (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 Q o site of the Cyt b 6 f complex.

Much evidence indicates that superoxide is generated from O 2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O 2 , little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H 2 O 2 -peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H 2 O 2 , which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H 2 O 2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O 2 . Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O 2 . These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O 2 to O 2 - ., (Copyright © 2022. Published by Elsevier Inc.)

The cytochrome bc 1 complex is an essential component of the mitochondrial respiratory chain of the yeast Saccharomyces cerevisiae. It is composed of ten protein subunits, three of them playing an important role in electron transfer and proton pumping across the inner mitochondrial membrane. Cytochrome b , the central component of this respiratory complex, is encoded by the mitochondrial genome, whereas all the other subunits are of nuclear origin. The assembly of all these subunits into the mature and functional cytochrome bc 1 complex is therefore a complicated process which requires the participation of several chaperone proteins. It has been found that the assembly process of the mitochondrial bc 1 complex proceeds through the formation of distinct sub-complexes in an ordered sequence. Most of these sub-complexes have been thoroughly characterized, and their molecular compositions have also been defined. This study critically analyses the results obtained so far and highlights new possible areas of investigation.

Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit photosystem II complex which functions in oxygenic photosynthesis, there is presently no consensus on the function of b-559 in the photosynthetic electron transport chain. The inability in earlier times to define a redox-linked function of this cytochrome was, to a large extent, a consequence of an absence of biochemical and structure information to complement an extensive array of spectrophotometric studies of the cytochrome in situ. Based on the location of hetero-dimeric b-559 in the photosystem II reaction center complex, derived from crystal crystallographic structure analysis, and the absence of a necessary redox function for the cytochrome in PSII, it is proposed that the main function of cytochrome b-559 is linked to its role as a structure component in the PSII reaction center complex. This function resides in the association of b-559 through its heme histidine residues in the trans-membrane domains of the PsbE and PsbF subunits of the PSII reaction center. These subunits, along with PsbJ, are inferred, from the analysis of structure, to define the intra-membrane portal in the PSII reaction center for plastoquinol (PQH 2 ) export which, through the PSII complex, provides the redox link to the cytochrome b 6 f complex in the electron transfer chain., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

We recently reported that the membrane-associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b 5 (cytb 5 ) domain proteins, called cytb 5M (MAPR-like). Relative to classical cytb 5 proteins, MAPR and ctyb 5M proteins shared unique sequence elements and a distinct heme-binding orientation at an approximately 90° rotation relative to classical cytb 5 , as demonstrated in the archetypal crystal structure of a cytb 5M protein (PDB accession number 6NZX). Here, we present the crystal structure of an archaeal cytb 5M domain (Methanococcoides burtonii WP&#95;011499504.1, PDB:6VZ6). It exhibits similar heme binding to the 6NZX cytb 5M , supporting the deduction that MAPR-like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The conversion of light energy to chemical energy by photosynthesis requires the concerted action of large protein complexes in the thylakoid membrane. Recent work has provided fundamental insights into the three-dimensional structure of these complexes, but how they are assembled from hundreds of parts remains poorly understood. Particularly little is known about the biogenesis of the cytochrome b 6 f complex (Cytb 6 f), the redox-coupling complex that interconnects the two photosystems. Here we report the identification of a factor that guides the assembly of Cytb 6 f in thylakoids of chloroplasts. The protein, DE-ETIOLATION-INDUCED PROTEIN 1 (DEIP1), resides in the thylakoid membrane and is essential for photoautotrophic growth. Knock-out mutants show a specific loss of Cytb 6 f, and are defective in complex assembly. We demonstrate that DEIP1 interacts with the two cytochrome subunits of the complex, PetA and PetB, and mediates the assembly of intermediates in Cytb 6 f biogenesis. The identification of DEIP1 provides an entry point into the study of the assembly pathway of a crucial complex in photosynthetic electron transfer., (© 2022. The Author(s).)

Cytochrome P450 monooxygenases (P450s) are well studied enzymes catalyzing the oxidative metabolism of xenobiotics in insects including mosquitoes. Their duplication and upregulation in agricultural and public health pests such as anopheline mosquitoes often leads to an enhanced metabolism of insecticides which confers resistance. In the laboratory strain Anopheles funestus FUMOZ-R the duplicated P450s CYP6P9a and CYP6P9b are highly upregulated and proven to confer pyrethroid resistance. Microsomal P450 activity is regulated by NADPH cytochrome P450 oxidoreductase (CPR) required for electron transfer, whereas the modulatory role of cytochrome b 5 (CYB5) on insect P450 activity is less clear. In previous studies CYP6P9a and CYP6P9b were recombinantly expressed in tandem with An. gambiae CPR using E. coli-expression systems and CYB5 added to the reaction mix to enhance activity. However, the precise role of CYB5 on substrate turn-over when combined with CYP6P9a and CYP6P9b remains poorly investigated, thus one objective of our study was to address this knowledge gap. In contrast to the CYP6P9 variants, the expression levels of both CYB5 and CPR were not upregulated in the pyrethroid resistant FUMOZ-R strain when compared to the susceptible FANG strain, suggesting no immediate regulatory role of these genes in pyrethroid resistance in FUMOZ-R. Here, for the first time we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus in a baculovirus expression system using High-5 insect cells. Co-expression of each enzyme with CPR from either An. gambiae or An. funestus did not reveal noteworthy differences in catalytic capacity. Whereas the co-expression of An. funestus CYB5 - tested at different multiplicity of infection (MOI) ratios - resulted in a significantly higher metabolization of coumarin substrates as measured by fluorescence assays. This was confirmed by Michaelis-Menten kinetics using the most active substrate, 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC). We observed a similar increase in coumarin substrate turnover by adding human CYB5 to the reaction mix. Finally, we compared by UPLC-MS/MS analysis the depletion rate of deltamethrin and the formation of 4'OH-deltamethrin by recombinantly expressed CYP6P9a and CYP6P9b with and without CYB5 and detected no difference in the extent of deltamethrin metabolism. Our results suggest that co-expression (or addition) of CYB5 with CYP6P9 variants, recombinantly expressed in insect cells, can significantly enhance their metabolic capacity to oxidize coumarins, but not deltamethrin., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Neutrophils play a very key role in the human immune defense against pathogenic infections. The predominant players in this role during the activation of neutrophils are the release of cytotoxic agents stored in the granules and secretory vesicles and the massive production of reactive oxygen species (ROS) initiated by the enzyme NADPH oxidase. In addition, in living organisms, cells are continuously exposed to endogenous (inflammations, elevated neutrophil presence in the vicinity) and exogenous ROS at low and moderate levels (travels by plane, radiotherapy, space irradiation, blood banking, etc.). To study these effects, we used ROS induced by gamma radiation from low (0.2 Gy) to high (25 Gy) dose levels on PLB-985 cells from a myeloid cell line differentiated to neutrophil-like cells that are considered a good alternative to neutrophils. We determined a much longer lifetime of PLB-985 cells than that of neutrophils, which, as expected, decreased by increasing the irradiation dose. In the absence of any secondary stimulus, a very low production of ROS is detected with no significant difference between irradiated and non-irradiated cells. However, in phagocytosing cells, irradiation doses above 2 Gy enhanced oxidative burst in PLB-985 cells. Whatever the irradiation dose, NADPH oxidase devoid of its cytosolic regulatory units is observed at the plasma membrane in irradiated PLB-985 cells. This result is different from that observed for irradiated neutrophils in which irradiation also induced a translocation of regulatory subunits suggesting that the signal transduction mechanism or pathway operate differently in both cells.

Cytochrome (Cyt) b 559 is a key component of the photosystem II complex (PSII) that is essential for its proper functioning and assembly. Site-directed mutants of the model cyanobacterium Synechocystis sp. PCC6803 with mutated heme axial ligands of Cyt b 559 have little PSII and are therefore unable to grow photoautotrophically. Here we describe two types of Synechocystis autotrophic transformants that retained the same mutations in Cyt b 559 but are able to accumulate PSII and grow photoautotrophically. Whole-genome sequencing revealed that all of these autotrophic transformants carried a variable number of tandem repeats (from 5 to 15) of chromosomal segments containing the psbEFLJ operon. RNA-seq analysis showed greatly increased transcript levels of the psbEFLJ operon in these autotrophic transformants. Multiple copies of the psbEFLJ operon in these transformants were only maintained during autotrophic growth, while its copy numbers gradually decreased under photoheterotrophic conditions. Two-dimensional PAGE analysis of membrane proteins revealed a strong deficiency in PSII complexes in the Cyt b 559 mutants that was reversed in the autotrophic transformants. These results illustrate how tandem gene amplification restores PSII accumulation and photoautotrophic growth in Cyt b 559 mutants of cyanobacteria, and may serve as an important adaptive mechanism for cyanobacterial survival., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

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 b 5 reductase (CBR) and/or cytochrome b 5 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., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Potassium (K + ) is an essential element for growth and development in both animals and plants, while high levels of environmental sodium (Na + ) represent a threat to most plants. The uptake of K + from high-saline environments is an essential mechanism to maintain intracellular K + /Na + homeostasis, which can help reduce toxicity caused by Na + accumulation, thereby improving the salt tolerance of plants. However, the mechanisms and regulation of K + -uptake during salt stress remain poorly understood. In this study, we identified an endoplasmic reticulum-localized cytochrome b 5 (OsCYB5-2) that interacted with a high-affinity K + transporter (OsHAK21) at the plasma membrane. The association of OsCYB5-2 with the OsHAK21 transporter caused an increase in transporter activity by enhancing the apparent affinity for K + -binding but not Na + -binding. Heme binding to OsCYB5-2 was essential for the regulation of OsHAK21. High salinity directly triggered the OsHAK21-OsCYB5-2 interaction, promoting OsHAK21-mediated K + -uptake and restricting Na + entry into cells; this maintained intracellular K + /Na + homeostasis in rice cells. Finally, overexpression of OsCYB5-2 increased OsHAK21-mediated K + transport and improved salt tolerance in rice seedlings. This study revealed a posttranslational regulatory mechanism for HAK transporter activity mediated by a cytochrome b 5 and highlighted the coordinated action of two proteins to perceive Na + in response to salt stress., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Mitochondrial mRNAs encode key subunits of the oxidative phosphorylation complexes that produce energy for the cell. In Saccharomyces cerevisiae, mitochondrial translation is under the control of translational activators, specific to each mRNA. In Schizosaccharomyces pombe, which more closely resembles the human system by its mitochondrial DNA structure and physiology, most translational activators appear to be either lacking, or recruited for post-translational functions. By combining bioinformatics, genetic and biochemical approaches we identified two interacting factors, Cbp7 and Cbp8, controlling Cytb production in S. pombe. We show that their absence affects cytb mRNA stability and impairs the detection of the Cytb protein. We further identified two classes of Cbp7/Cbp8 partners and showed that they modulated Cytb or Cox1 synthesis. First, two isoforms of bS1m, a protein of the small mitoribosomal subunit, that appear mutually exclusive and confer translational specificity. Second, a complex of four proteins dedicated to Cox1 synthesis, which includes an RNA helicase that interacts with the mitochondrial ribosome. Our results suggest that S. pombe contains, in addition to complexes of translational activators, a heterogeneous population of mitochondrial ribosomes that could specifically modulate translation depending on the mRNA translated, in order to optimally balance the production of different respiratory complex subunits., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Insight into mammalian respiratory complexes defines the role of allosteric protein interactions in their proton-motive activity. In cytochrome c oxidase (CxIV) conformational change of subunit I, caused by O 2 binding to heme a 3 2+ -Cu B + and reduction, and stereochemical transitions coupled to oxidation/reduction of heme a and Cu A , combined with electrostatic effects, determine the proton pumping activity. In ubiquinone-cytochrome c oxidoreductase (CxIII) conformational movement of Fe-S protein between cytochromes b and c 1 is the key element of the proton-motive activity. In NADH-ubiquinone oxidoreductase (CxI) ubiquinone binding and reduction result in conformational changes of subunits in the quinone reaction structure which initiate proton pumping., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Sylvatic populations of Triatoma infestans represent a challenge to Chagas disease control as they are not targeted by vector control activities and may play a key role in post-spraying house re-infestation. Understanding sylvatic foci distribution and gene flow between sylvatic and domestic populations is crucial to optimize vector control interventions and elucidate the development and spread of insecticide resistance. Herein, the genetic profiles of five Andean T. infestans populations from Bolivia with distinct insecticide susceptibility profiles were compared. Multilocus genotypes based on eight microsatellites and the DNA sequence of a fragment of the cytochrome B (cytB) gene were obtained for 92 individuals. CytB haplotypes were analyzed with previously reported Bolivian T. infestans haplotypes to evaluate putative historical gene flow among populations. Each specimen was also screened for two nucleotide mutations in the sodium channel gene (kdr), related to pyrethroid resistance (L1014 and L9251). Significant genetic differentiation was observed among all populations, although individuals of admixed origin were detected in four of them. Notably, the genetic profiles of adjacent domestic and sylvatic populations of Mataral, characterized by higher levels of insecticide resistance, support their common ancestry. Only one sylvatic individual from Mataral carried the kdr mutation L1014, suggesting that this mechanism is unlikely to cause the altered insecticide susceptibility observed in these populations. However, as the resistance mutation is present in the area, it has the potential to be selected under insecticidal pressure. Genetic comparisons of these populations suggest that insecticide resistance is likely conferred by ancient trait(s) in T. infestans sylvatic populations, which are capable of invading domiciles. These results emphasize the need for stronger entomological surveillance in the region, including early detection of house invasion, particularly post-spraying, monitoring for resistance to pyrethroids and the design of integrative control actions that consider sylvatic foci around domestic settings and their dispersion dynamics., (Published by Elsevier B.V.)

Complexome Profiling (CP) combines size separation, by electrophoresis or other means, of native multimeric complexes with protein identification by mass spectrometry (MS). Peptide MS analysis of the multiple fractions in which the sample is separated, results in the creation of protein abundance profiles in function of molecular size, providing a visual output of the assembly status of a group of proteins of interest. Stable isotope labeling by amino acids in cell culture (SILAC) is an established quantitative proteomics technique that allows duplexing in the MS analysis as well as the comparison of relative protein abundances between the samples, which are processed and analyzed together. Combining SILAC and CP permitted the direct comparison of migration and abundance of the proteins present in the mitochondrial respiratory chain complexes in two different samples. This analysis, however, introduced a level of complexity in data processing for which bioinformatic tools had to be developed in order to generate the normalized protein abundance profiles. The advantages and challenges of using of this type of analysis for the characterization of two cell lines carrying pathological variants in MT-CO3 and MT-CYB is reviewed. An additional unpublished example of SILAC-CP of a cell line with an in-frame 18-bp deletion in MT-CYB is presented. In these cells, in contrast to other MT-CYB deficient models, a small proportion of complex III 2 is formed and it is found associated with fully assembled complex I. This analysis also revealed a profuse accumulation of assembly intermediates containing complex III subunits UQCR10 and CYC1, as well as a profound early-stage complex IV assembly defect., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Iron supplements are limited by their poor absorption and low efficacy. A circadian feeding schedule would affect the circadian rhythm and improve nutrient metabolism. In this study, 18 iron-deficient piglets were randomly assigned to three groups: a control group receiving a constant diet with mid-iron (MI), a 'HL' group receiving a high-iron (HI) diet at 8:00 h and a low-iron (LI) diet at 18:00, and an 'LH' group receiving a LI diet at 8:00 and a HI diet at 18:00. The effects of circadian iron administration on iron absorption, iron status, and biological rhythm in iron-deficient piglets were investigated., Results: Serum iron and hemoglobin improved significantly (P < 0.05) but did not significantly differ in the circadian iron-feeding groups (P > 0.05). Iron concentration in the liver and spleen was significantly higher in the LH group than in the HL group (P < 0.05), and mRNA expression of divalent metal transport 1 (DMT1), cytochrome B (CYBRD1) and ferroportin (FPN) genes in the duodenum was significantly elevated in the LH group (P < 0.05). The clock-related genes showed differential expression in the duodenum, with greater mRNA expression for period (Per2) and cryptochrome (Cry1 and Cry2) in the LH group (P < 0.05)., Conclusion: Circadian iron administration affected iron absorption and iron storage in pigs. Iron supplementation in the evening might be a more effective pattern for iron utilization. The rhythmic system in the intestine, driven by the time, played an important role in this process. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

The bc 1 complex is a proton pump of the mitochondrial electron transport chain which transfers electrons from ubiquinol to cytochrome c. It operates via the modified Q cycle in which the two electrons from oxidation of ubiquinol at the Q o center are bifurcated such that the first electron is passed to Cytc via an iron sulfur center and c 1 whereas the second electron is passed across the membrane by b L and b H to reduce ubiquinone at the Q i center. Proton pumping occurs because oxidation of ubiquinol at the Q o center releases protons to the P-side and reduction of ubiquinone at the Q i center takes up protons from the N-side. However, the mechanisms which prevent the thermodynamically more favorable short circuit reactions and so ensure precise bifurcation and proton pumping are not known. Here we use statistical thermodynamics to show that reaction steps that originate from high energy states cannot support high flux even when they have large rate constants. We show how the chemistry of ubiquinol oxidation and the structure of the Q o site can result in free energy profiles that naturally suppress flux through the short circuit pathways while allowing high rates of bifurcation. These predictions are confirmed through in-silico simulations using a Markov state model., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The paper shows that natural α,ω-dioic acid, α,ω-hexadecanedioic acid (HDA), is able to stimulate the respiration of succinate-fueled rat liver mitochondria in state 4 without induction of proton conductivity of the inner membrane. This effect of HDA is less pronounced in glutamate/malate-fueled mitochondria, as well as in the case of ascorbate/TMPD or ascorbate/ferrocyanide substrate systems, which transfer electrons directly to cytochrome c. It is noted that HDA-induced stimulation of respiration is not associated with damage to the inner membrane in a part of mitochondria and with shunting of electrons through the bc 1 complex. Therefore, HDA can be considered as a natural decoupling agent. Specific inhibitors of the bc 1 complex (antimycin A and myxothiazole) as well as malonate and dithionitrobenzoate were used in the inhibitory analysis. These and other experiments have shown that during the oxidation of succinate in liver mitochondria, the decoupling effect of HDA is mainly carried out at the level of the bc 1 complex. We hypothesized that HDA is capable of promoting the cyclic transport of protons within the bc 1 complex and thus switch this complex to the idle mode of operation (intrinsic uncoupling of the bc 1 complex). Induction of free respiration in liver mitochondria by HDA at the level of the bc 1 complex is considered as one of the "rescue pathways" of hepatocytes in various pathological conditions, accompanied by disorders of carbohydrate and lipid metabolism and increased oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Proteins have been used as building blocks to provide various supramolecular structures in efforts to develop nano-biomaterials possessing broad biological functionalities. A series of unique structures have been obtained from the engineering of hemoproteins which contain the iron porphyrin known as heme, as a prosthetic group. This work in developing assembling systems is extended using cytochrome b 562 , a small electron transfer hemoprotein engineered to include an externally-attached heme moiety. The engineered units, which form a one-dimensional assembly via interprotein heme-heme pocket interactions, are conjugated to an apo-form of hexameric tyrosine-coordinated hemoprotein (apoHTHP) to provide a branching unit promoting the assembly of a star-shaped structure. The incorporation of the heme moiety attached to the protein surface of cytochrome b 562 into apoHTHP can be accelerated by elevating the reaction temperature to generate a new assembly. The formation of a new larger assembly structure was confirmed by size exclusion chromatography. The ratio of the heme-containing units in the assemblies was analyzed by UV-Vis spectroscopy and the population of protein units estimated from SDS PAGE suggests the presence of plausible star-shaped structures, which are supported by hydrodynamic diameter data obtained by dynamic light scattering.

The cytochrome bc 1 complex is a transmembrane enzymatic protein complex that plays a central role in cellular energy production and is present in both photosynthetic and respiratory chain organelles. Its reaction mechanism is initiated by the binding of a quinol molecule to an active site, followed by a series of charge transfer reactions between the quinol and protein subunits. Previous work hypothesized that the primary reaction was a concerted proton-coupled electron transfer (PCET) reaction because of the apparent absence of intermediate states associated with single proton or electron transfer reactions. In the present study, the kinetics of the primary bc 1 complex PCET reaction is investigated with a vibronically nonadiabatic PCET theory in conjunction with all-atom molecular dynamics simulations and electronic structure calculations. The computed rate constants and relatively high kinetic isotope effects are consistent with experimental measurements on related biomimetic systems. The analysis implicates a concerted PCET mechanism with significant hydrogen tunneling and nonadiabatic effects in the bc 1 complex. Moreover, the employed theoretical framework is shown to serve as a general strategy for describing PCET reactions in bioenergetic systems.