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Silver (Ag) is a pivotal transition metal with applications in multiple industries, necessitating efficient recovery techniques. Despite various proposed methods for silver recovery from wastewaters, challenges persist especially for low concentrations. In this context, bioreduction by bacteria like Geobacter sulfurreducens, offers a promising approach by converting Ag(I) to Ag nanoparticles. To reveal the mechanisms driving microbial Ag(I) reduction, we conducted transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition. Integrated transcriptomic and protein-protein interaction network analyses identified significant transcriptional shifts, predominantly linked to c-type cytochromes, NADH, and pili. When compared to a pilus-deficient strain, the wild-type strain exhibited distinct cytochrome gene expressions, implying specialized functional roles. Additionally, despite a down-regulation in NADH dehydrogenase genes, we observed up-regulation of specific downstream cytochrome genes, highlighting NADH's potential role as an electron donor in the Ag(I) reduction process. Intriguingly, our findings also highlight the significant influence of pili on the morphology of the resulting Ag nanoparticles. The presence of pili led to the formation of smaller and more crystallized Ag nanoparticles. Overall, our findings underscore the intricate interplay of cytochromes, NADH, and pili in Ag(I) reduction. Such insights suggest potential strategies for further enhancing microbial Ag(I) reduction., 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 Ltd. All rights reserved.)

C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production of c-type cytochromes in Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation of heme c are only expressed under anaerobic conditions. Different strategies were devised to bypass this obstacle, such as co-expressing the ccm genes from the pEC86 vector. However, co-expression methods restrict the choice of expression host and vector. Here we describe the first use of Vibrio natriegens V max X2 for the recombinant production of difficult-to-express redox proteins from the extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three c-type cytochromes. Co-expression of the ccm genes was not required to produce holo-c-type cytochromes in V max X2. E. coli T7 Express only produced holo-c-type cytochromes during co-expression of the ccm genes and was not able to produce the inner membrane cytochrome CycA. Additionally, V max X2 cell extracts contained higher portions of recombinant holo-proteins than T7 Express cell extracts. All redox proteins were translocated to the intended cell compartment in both hosts. In conclusion, V. natriegens represents a promising alternative for the production of c-type cytochromes and difficult-to-express redox proteins., (© 2024. The Author(s).)

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to delineate the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

This article explores the potential of cytochromes P450 (P450s) as biocatalysts for oxidation reactions. Despite their potential, P450s have not been extensively used in research and industry due to challenges. The authors present a case study on the iron-catalyzed Wacker-type oxidation of olefins using ambient air as the sole oxidant, which has shown promising results for the functionalization of complex molecules. The text also discusses other strategies for the oxidation of organic compounds to produce aryl aldehydes, highlighting their selectivity and compatibility with complex molecules. The article concludes by discussing recent advances in catalysis inspired by P450 enzymes, which have the potential to accelerate the development of versatile synthetic catalysts with applications in drug synthesis and bioremediation. [Extracted from the article]

Lenvatinib is an orally effective tyrosine kinase inhibitor used to treat several types of tumors, including progressive, radioiodine-refractory differentiated thyroid cancer and advanced renal cell carcinoma. Although this drug is increasingly used in therapy, its metabolism and effects on the organism are still not described in detail. Using the rat as an experimental animal model, this study aimed to investigate the metabolism of lenvatinib by rat microsomal enzymes and cytochrome P450 (CYPs) enzymes recombinantly expressed in SupersomesTM in vitro and to assess the effect of lenvatinib on rat CYP expression in vivo. Two metabolites, O-desmethyl lenvatinib, and lenvatinib N-oxide, were produced by rat CYPs in vitro. CYP2A1 and 2C12 were found to be the most effective in forming O-desmethyl lenvatinib, while CYP3A2 was found to primarily form lenvatinib N-oxide. The administration of lenvatinib to rats caused changes in the expression of mRNA and protein, as well as the activity of various CYPs, particularly in an increase in CYP1A1. Thus, the administration of lenvatinib to rats has an impact on the level of CYPs.

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug–drug interactions involving IP6 are low. [ABSTRACT FROM AUTHOR]

Bacteria utilize electron conduction in their communities to drive their metabolism, which has led to the development of various environmental technologies, such as electrochemical microbial systems and anaerobic digestion. It is challenging to measure the conductivity among bacterial cells when they hardly form stable biofilms on electrodes. This makes it difficult to identify the biomolecules involved in electron conduction. In the present study, we aimed to identify c -type cytochromes involved in electron conduction in Shewanella oneidensis MR-1 and examine the molecular mechanisms. We established a colony-based bioelectronic system that quantifies bacterial electrical conductivity, without the need for biofilm formation on electrodes. This system enabled the quantification of the conductivity of gene deletion mutants that scarcely form biofilms on electrodes, demonstrating that c -type cytochromes, MtrC and OmcA, are involved in electron conduction. Furthermore, the use of colonies of gene deletion mutants demonstrated that flavins participate in electron conduction by binding to OmcA, providing insight into the electron conduction pathways at the molecular level. Furthermore, phenazine-based electron transfer in Pseudomonas aeruginosa PAO1 and flavin-based electron transfer in Bacillus subtilis 3610 were confirmed, indicating that this colony-based system can be used for various bacteria, including weak electricigens.

Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>10 6  s -1 ) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<10 5  s -1 ) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance., (© 2024. The Author(s).)

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease. [ABSTRACT FROM AUTHOR]

Abstract C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production of c-type cytochromes in Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation of heme c are only expressed under anaerobic conditions. Different strategies were devised to bypass this obstacle, such as co-expressing the ccm genes from the pEC86 vector. However, co-expression methods restrict the choice of expression host and vector. Here we describe the first use of Vibrio natriegens Vmax X2 for the recombinant production of difficult-to-express redox proteins from the extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three c-type cytochromes. Co-expression of the ccm genes was not required to produce holo-c-type cytochromes in Vmax X2. E. coli T7 Express only produced holo-c-type cytochromes during co-expression of the ccm genes and was not able to produce the inner membrane cytochrome CycA. Additionally, Vmax X2 cell extracts contained higher portions of recombinant holo-proteins than T7 Express cell extracts. All redox proteins were translocated to the intended cell compartment in both hosts. In conclusion, V. natriegens represents a promising alternative for the production of c-type cytochromes and difficult-to-express redox proteins.

Abstract Anaerobic microbial corrosion of iron‐containing metals causes extensive economic damage. Some microbes are capable of direct metal‐to‐microbe electron transfer (electrobiocorrosion), but the prevalence of electrobiocorrosion among diverse methanogens and acetogens is poorly understood because of a lack of tools for their genetic manipulation. Previous studies have suggested that respiration with 316L stainless steel as the electron donor is indicative of electrobiocorrosion, because, unlike pure Fe0, 316L stainless steel does not abiotically generate H2 as an intermediary electron carrier. Here, we report that all of the methanogens (Methanosarcina vacuolata, Methanothrix soehngenii, and Methanobacterium strain IM1) and acetogens (Sporomusa ovata and Clostridium ljungdahlii) evaluated respired with pure Fe0 as the electron donor, but only M. vacuolata, Mx. soehngenii, and S. ovata were capable of stainless steel electrobiocorrosion. The electrobiocorrosive methanogens required acetate as an additional energy source in order to produce methane from stainless steel. Cocultures of S. ovata and Mx. soehngenii demonstrated how acetogens can provide acetate to methanogens during corrosion. Not only was Methanobacterium strain IM1 not capable of electrobiocorrosion, but it also did not accept electrons from Geobacter metallireducens, an effective electron‐donating partner for direct interspecies electron transfer to all methanogens that can directly accept electrons from Fe0. The finding that M. vacuolata, Mx. soehngenii, and S. ovata are capable of electrobiocorrosion, despite a lack of the outer‐surface c‐type cytochromes previously found to be important in other electrobiocorrosive microbes, demonstrates that there are multiple microbial strategies for making electrical contact with Fe0.

Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we use cryo-EM and perform steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III 2 -IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Negatively charged loops of CIII 2 subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allows us to identify water molecules in proton pathways of CIV and, to the best of our knowledge, previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII 2 loops to conduct electron transfer between CIII 2 and CIV., (© 2024. The Author(s).)

Mass spectrometry (MS) using an electron multiplier for intact protein analysis remains limited. Because of the massive size and complex structure of proteins, the slow flight speed of their ions results in few secondary electrons and thus low detection sensitivity and poor spectral resolution. Thus, we present a compact ion trap-mass spectrometry approach to directly detect ion packets and obtain the high-resolution molecular signature of proteins. The disturbances causing deviations of ion motion and mass conversion have been clarified in advance. The radio frequency waveform used to manipulate ions is proposed to be a sequence of constant-frequency steps, interconnected by short time-outs, resulting in least dispersive distortion. Furthermore, more such constant-phase conjunctions are arranged in each step to compensate for fluctuations resulting from defects in the system and operation. In addition, two auxiliary pulses are generated in the right phase of each step to select ions of a specific secular state to detect one clean and sharp spectral line.This study demonstrates a top-down approach for the MS measurement of cytochrome C molecules, resulting in a spectral profile of the protein in its natural state at a resolution of 20 Da. Additionally, quick MS scans of other proteins were performed.

To investigate their roles in extracellular electron transfer (EET), the porin-cytochrome ( pcc ) gene clusters Gmet0825-0828 , Gmet0908-0910 , and Gmet0911-0913 of the Gram-negative bacterium Geobacter metallireducens were deleted. Failure to delete all pcc gene clusters at the same time suggested their essential roles in extracellular reduction of Fe(III)-citrate by G. metallireducens . Deletion of Gmet0825-0828 had no impact on bacterial reduction of Fe(III)-citrate but diminished bacterial reduction of ferrihydrite and abolished anode reduction and direct interspecies electron transfer (DIET) to Methanosarcina barkeri and Geobacter sulfurreducens . Although it had no impact on the bacterial reduction of Fe(III)-citrate, deletion of Gmet0908-0910 delayed ferrihydrite reduction, abolished anode reduction, and diminished DIET. Deletion of Gmet0911-0913 had little impact on DIET but diminished bacterial reductions of Fe(III)-citrate, ferrihydrite, and anodes. Most importantly, deletions of both Gmet0825-0828 and Gmet0908-0910 restored bacterial reduction of ferrihydrite and anodes and DIET. Enhanced expression of Gmet0911-0913 in this double mutant when grown in coculture with G. sulfurreducens Δ hybL Δ fdnG suggested that this cluster might compensate for impaired EET functions of deleting Gmet0825-0828 and Gmet0908-0910 . Thus, these pcc gene clusters played essential, distinct, overlapping, and compensatory roles in EET of G. metallireducens that are difficult to characterize as deletion of some clusters affected expression of others. The robustness of these pcc gene clusters enabled G. metallireducens to mediate EET to different acceptors for anaerobic growth even when two of its three pcc gene clusters were inactivated by mutation. The results from this investigation provide new insights into the roles of pcc gene clusters in bacterial EET., Importance: The Gram-negative bacterium Geobacter metallireducens is of environmental and biotechnological significance. Crucial to the unique physiology of G. metallireducens is its extracellular electron transfer (EET) capability. This investigation sheds new light on the robust roles of the three porin-cytochrome ( pcc ) gene clusters, which are directly involved in EET across the bacterial outer membrane, in the EET of G. metallireducens . In addition to their essential roles, these gene clusters also play distinct, overlapping, and compensatory roles in the EET of G. metallireducens . The distinct roles of the pcc gene clusters enable G. metallireducens to mediate EET to a diverse group of electron acceptors for anaerobic respirations. The overlapping and compensatory roles of the pcc gene clusters enable G. metallireducens to maintain and restore its EET capability for anaerobic growth when one or two of its three pcc gene clusters are deleted from the genome., Competing Interests: The authors declare no conflict of interest.

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 .

This article describes the molecular mechanism by which tetraalkylammonium chloride ([R 4 N]Cl: R- = methyl (Me), ethyl (Et), propyl (Pr),butyl (Bu)) modulates the stability, folding, and dynamics of cytochrome c (Cyt c). Analysis of [R 4 N]Cl effects on thermal/chemical denaturations, millisecond refolding/unfolding kinetics, and slow CO-association kinetics of Cyt c without and with denaturant providing some significant results: (i) [R 4 N]Cl decreasing the unfolding free energy estimated by thermodynamic and kinetic analysis of thermal/chemical denaturation curves and kinetic chevrons (Log k obs -[GdmCl]) of Cyt c, respectively (ii) hydrophobicity of R-group of [R 4 N]Cl, preferential inclusion of [R 4 N]Cl at the protein surface, and destabilizing enthalpic attractive interactions of [Me 4 N]Cl and steric entropic interactions of [Et 4 N]Cl,[Pr 4 N]Cl and [Bu 4 N]Cl with protein contribute to [R 4 N]Cl-induced decrease thermodynamic stability of Cyt c (iii) [R 4 N]Cl exhibits an additive effect with denaturant to decrease thermodynamic stability and refolding rates of Cyt c (iv) low concentrations of [R 4 N]Cl (≤ 0.5 M) constrain the motional dynamics while the higher concentrations (>0.75 M [R 4 N]Cl) enhance the structural-fluctuations that denture protein (v) hydrophobicity of R-group of [R 4 N]Cl alters the [denaturant]-dependent conformational stability, refolding-unfolding kinetics, and CO-association kinetics of Cyt c. Furthermore, the MD simulations depicted that the addition of 1.0 M of [R 4 N]Cl increased the conformational fluctuations in Cyt c leading to decreased structural stability in the order [Me 4 N]Cl < [Et 4 N]Cl < [Pr 4 N]Cl < [Bu 4 N]Cl consistent with the experimental results., 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. All rights reserved.)

Cytochrome c (cyt c) has been found to play a function in apoptosis in cell-free models. This work presents the creation of molecularly imprinted conducting poly(3, 4-ethylenedioxythiopene) (MIPEDOT) on the surface of a screen printed carbon electrode (SPCE) for cyt c. Cyt c was imprinted by electropolymerization due to the presence of an EDOT monomer hydrophobic functional group on SPCE, using CV to obtain highly selective materials with excellent molecular recognition ability. MIPEDOT was characterized by CV, EIS, and DPV using ferricyanide/ferrocyanide as a redox probe. Further, the characterization of the sensor was accomplished using SEM for surface morphological confirmation. Using CV, the peak current measured at the potential of +1 to -1 V (vs. Ag/AgCl) is linear in the cyt c concentration range from 1 to 1200 pM, showing a remarkably low detection limit of 0.5 pM (sensitivity:0.080 μA pM). Moreover, the applicability of the approach was successfully confirmed with the detection of cyt c in biological samples (human plasma). Similarly, our research has proven a low-cost, simple, and efficient sensing platform for cyt c detection, rendering it a viable tool for the future improvement of reliable and exact non-encroaching cell death detection., 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 hydrogen/deuterium (H/D) exchange rate is an optimal measure for studying the structures and dynamics of hydrogen bonding systems, as it reflects the molecular contact environment and the strength of the hydrogen bonds. A method for rapid measurement of the H/D exchange reaction rates is required to examine the intermolecular environments of molecules in solutions. We developed a droplet collision atmospheric pressure infrared laser ablation mass spectrometry technique for this purpose. We obtained the H/D exchange reaction rate of cytochrome c in a methanol/H 2 O·D 2 O solution. We revealed that the first hydration shell of the cytochrome c molecule hinders the penetration of D 2 O to the surface of the molecule from the rates, which provides a novel method to investigate solution structures by a mass-spectrometric method. The droplet-collision mass spectrometry method developed in the present study can be extended to research on the molecular interactions in solutions, such as the mutual interactions of protein molecules, which are of importance in living cells.

Nanopore sensing is a label-free single-molecule technique that enables the study of the dynamical structural properties of proteins. Here, we detect the translocation of cytochrome c (Cyt c ) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt c conformation during its translocation in an electric field. Before Cyt c translocates through an asymmetric thin SiN x nanopore, ∼1 ms trapping events occur due to electric field-induced denaturation. These trapping events were corroborated by a control analysis with a transmission electron microscopy-drilled pore and denaturant buffer. Cyt c translocation events exhibited markedly greater broad current blockade when the pores were photothermally heated. Collectively, our molecular dynamics simulation predicted that an increased temperature facilitates denaturation of the α-helical structure of Cyt c , resulting in greater blockade current during Cyt c trapping. Our photothermal heating method can be used to study the influence of temperature on protein conformation at the single-molecule level in a label-free manner.

Cytochromes c'-α are nitric oxide (NO)-binding heme proteins derived from bacteria that can thrive in a wide range of temperature environments. Studies of mesophilic Alcaligenes xylosoxidans cytochrome c'-α (AxCP-α) have revealed an unusual NO-binding mechanism involving both heme faces, in which NO first binds to form a distal hexa-coordinate Fe(II)-NO (6cNO) intermediate and then displaces the proximal His to form a proximal penta-coordinate Fe(II)-NO (5cNO) final product. Here, we characterize a thermally stable cytochrome c'-α from thermophilic Hydrogenophilus thermoluteolus (PhCP-α) to understand how protein thermal stability affects NO binding. Electron paramagnetic and resonance Raman spectroscopies reveal the formation of a PhCP-α 5cNO product, with time-resolved (stopped-flow) UV-vis absorbance indicating the involvement of a 6cNO intermediate. Relative to AxCP-α, the rates of 6cNO and 5cNO formation in PhCP-α are ∼11- and ∼13-fold lower, respectively. Notably, x-ray crystal structures of PhCP-α in the presence and absence of NO suggest that the sluggish formation of the proximal 5cNO product results from conformational rigidity: the Arg-132 residue (adjacent to the proximal His ligand) is held in place by a salt bridge between Arg-75 and Glu-135 (an interaction not present in AxCP-α or a psychrophilic counterpart). Overall, our data provide fresh insights into structural factors controlling NO binding in heme proteins, including 5cNO complexes relevant to eukaryotic NO sensors., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cytochrome c (CytC) is conjugated with a small molecule TG6 to give TG6-CytC, which is directly delivered into cytosol, triggering the release of endogenous CytC from mitochondria, and inducing a caspase-3-dependent apoptosis with an IC 50 down to 2.4 nM. This work shows an efficient strategy for intracellular protein delivery.

Background and Objectives : Most patients who are successfully resuscitated from cardiac arrest remain comatose, and only half regain consciousness 72 h after the arrest. Neuroprognostication methods can be complex and even inconclusive. As mitochondrial components have been identified as markers of post-cardiac-arrest injury and associated with survival, we aimed to investigate cytochrome c and mtDNA in comatose patients after cardiac arrest to compare neurological outcomes and to evaluate the markers' neuroprognostic value. Materials and Methods : This prospective observational study included 86 comatose post-cardiac-arrest patients and 10 healthy controls. Cytochrome c and mtDNA were determined at admission. Neuron-specific enolase (NSE) was measured after 72 h. Additional neuroprognostication methods were performed when patients remained unconscious. Cerebral performance category (CPC) was determined. Results : Cytochrome c was elevated in patients compared to healthy controls (2.029 [0.85-4.97] ng/mL vs. 0 [0.0-0.16], p < 0.001) but not mtDNA (95,228 [52,566-194,060] vs. 41,466 [28,199-104,708] copies/μL, p = 0.074). Compared to patients with CPC 1-2, patients with CPC 3-5 had higher cytochrome c (1.735 [0.717-3.40] vs. 4.109 [1.149-8.457] ng/mL, p = 0.011), with no differences in mtDNA (87,855 [47,598-172,464] vs. 126,452 [69,447-260,334] copies/μL, p = 0.208). Patients with CPC 1-2 and CPC 3-5 differed in all neuroprognostication methods. In patients with good vs. poor neurological outcome, ROC AUC was 0.664 ( p = 0.011) for cytochrome c, 0.582 ( p = 0.208) for mtDNA, and 0.860 ( p < 0.001) for NSE. The correlation between NSE and cytochrome c was moderate, with a coefficient of 0.576 ( p < 0.001). Conclusions : Cytochrome c was higher in comatose patients after cardiac arrest compared to healthy controls and higher in post-cardiac-arrest patients with poor neurological outcomes. Although cytochrome c correlated with NSE, its neuroprognostic value was poor. We found no differences in mtDNA.

Cytochrome c (Cyt-c) was commonly an intrinsic biomarker for a variety of cellular characteristics, such as respiration, energy levels, and apoptosis. Herein, a simple fluorescence sensor was constructed for the detection of Cyt-c in buffer and real serum samples. The carbon dots doped with Tb 3+ on the premise of 1-(2-pyridylazo)-2-naphthol (PAN) were fabricated and used as a dual-emission ratiometric fluorescent probe for detecting Cyt-c based on the internal filtering effect (IFE). As a fluorescent probe for ultra-sensitive detection, Cyt-c was quantitatively detected at different concentrations from 1 to 1000 nM. The fluorescent detection method for Cyt-c showed a good linear relationship from 1 to 50 nM, and the limit of detection (LOD) was 0.35 nM. In the recovery range of 101.27-103.39 % in human serum samples, the relative standard deviation (RSD) was less than 3.27 % (n = 3). In the end, the possible structures of CDs were predicted by DFT theoretical simulation calculations. All the results proved the ability of carbon dots as fluorescent probes to detect biomarkers and the application prospects in bioanalysis., 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. All rights reserved.)

Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4. In this study, we have investigated the binding of such variants and of wild type (wt) cyt C to synthetic cardiolipin-containing vesicles. The mutants have a lower propensity in membrane binding, displaying higher dissociation constants with respect to the wt protein. Compressibility measurements reveal that both variants are more flexible than the wt, suggesting that the native central Ω-loop is important for the interaction with membranes. Such hypothesis is supported by molecular dynamics simulations. A minimal distance analysis indicates that in the presence of cardiolipin the central Ω-loop of the mutants is no more in contact with the membrane, as it happens instead in the case of wt cyt C. Such finding might provide a hint for the reduced membrane binding capacity of the variants and their enhanced peroxidase activity in vivo., 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. Published by Elsevier B.V.)

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc 1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc 1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of C α atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H 2 O 2 and avoid complications of radical formation during the reaction.

Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S) lignin is lineage specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it coevolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in Helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs coopted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the reemergence of S-lignin biosynthesis in angiosperms., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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.)

AbstractCytochrome P40 (CYP) enzymes dominate the metabolism of numerous endogenous and xenobiotic substances. While it is commonly believed that CYP-catalysed reactions result in the detoxication of foreign substances, these reactions can also yield reactive intermediates that can bind to cellular macromolecules to cause cytotoxicity or irreversibly inactivate CYPs that create them.Mechanism-based inactivation (MBI) produces either irreversible or quasi-irreversible inactivation and is commonly caused by CYP metabolic bioactivation to an electrophilic reactive intermediate. Many drugs that have been known to cause MBI in CYPs have been discovered as perpetrators in drug-drug interactions throughout the last 20–30 years.This review will highlight the key findings from the recent literature about the mechanisms of CYP enzyme inhibition, with a focus on the broad mechanistic elements of MBI for widely used drugs linked to the phenomenon. There will also be a brief discussion of the clinical or pharmacokinetic consequences of CYP inactivation with regard to drug interaction and toxicity risk.Gaining knowledge about the selective inactivation of CYPs by common therapeutic drugs helps with the assessment of factors that affect the systemic clearance of co-administered drugs and improves comprehension of anticipated interactions with other drugs or xenobiotics.Cytochrome P40 (CYP) enzymes dominate the metabolism of numerous endogenous and xenobiotic substances. While it is commonly believed that CYP-catalysed reactions result in the detoxication of foreign substances, these reactions can also yield reactive intermediates that can bind to cellular macromolecules to cause cytotoxicity or irreversibly inactivate CYPs that create them.Mechanism-based inactivation (MBI) produces either irreversible or quasi-irreversible inactivation and is commonly caused by CYP metabolic bioactivation to an electrophilic reactive intermediate. Many drugs that have been known to cause MBI in CYPs have been discovered as perpetrators in drug-drug interactions throughout the last 20–30 years.This review will highlight the key findings from the recent literature about the mechanisms of CYP enzyme inhibition, with a focus on the broad mechanistic elements of MBI for widely used drugs linked to the phenomenon. There will also be a brief discussion of the clinical or pharmacokinetic consequences of CYP inactivation with regard to drug interaction and toxicity risk.Gaining knowledge about the selective inactivation of CYPs by common therapeutic drugs helps with the assessment of factors that affect the systemic clearance of co-administered drugs and improves comprehension of anticipated interactions with other drugs or xenobiotics. [ABSTRACT FROM AUTHOR]

Abstract Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106 s−1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (

The physiological role of Geobacter sulfurreducens extracellular cytochrome filaments is a matter of debate and the development of proposed electronic device applications of cytochrome filaments awaits methods for large-scale cytochrome nanowire production. Functional studies in G. sulfurreducens are stymied by the broad diversity of redox-active proteins on the outer cell surface and the redundancy and plasticity of extracellular electron transport routes. G. sulfurreducens is a poor chassis for producing cytochrome nanowires for electronics because of its slow, low-yield, anaerobic growth. Here we report that filaments of the G. sulfurreducens cytochrome OmcS can be heterologously expressed in Shewanella oneidensis. Multiple lines of evidence demonstrated that a strain of S. oneidensis, expressing the G. sulfurreducens OmcS gene on a plasmid, localized OmcS on the outer cell surface. Atomic force microscopy revealed filaments with the unique morphology of OmcS filaments emanating from cells. Electron transfer to OmcS appeared to require a functional outer-membrane porin-cytochrome conduit. The results suggest that S. oneidensis, which grows rapidly to high culture densities under aerobic conditions, may be suitable for the development of a chassis for producing cytochrome nanowires for electronics applications and may also be a good model microbe for elucidating cytochrome filament function in anaerobic extracellular electron transfer., (© 2024 Wiley Periodicals LLC.)

The current study aimed at population genetic characterization of B. vogeli based on the cytochrome b (cyt b) gene sequences (≥ 685 bp) available in the GenBank. Phylogenetic trees placed all the sequences of B. vogeli in a single large monophyletic clade; however, it was further divided into two subclades (Bv1 and Bv2). Out of seven nucleotide variations observed between Bv1 and Bv2 subclades, four were synonymous (G92A, C170T, T488C and A659G), and three were non-synonymous (G324A, C438A and G465A) resulting in amino acid substitutions at three places (V108I, L146I and V155I). Within different B. vogeli populations, the nucleotide and haplotype diversities were low. The median-joining haplotype network revealed only two haplotypes (Hap&#95;1 and Hap&#95;2). A geographical sub-structuring was noticed in the B. vogeli populations, with moderate genetic differentiation (F ST  = 0.05000; P < 0.05) and a very high gene flow (Nm = 4.75) between Indian and Chinese populations. Neutrality tests and mismatch distributions for the Indian population and the overall dataset of B. vogeli indicated a constant population size. This study provides the first insight into the genetic characterization, population genetics and haplotype network of B. vogeli based on the cyt b gene., (© 2024. The Author(s).)

Functional assays on intact tumor biopsies can complement genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key therapeutic response determinants, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Most of these assays rely on fluorescent labeling, a semiquantitative method best suited for single-time-point assays or labor-intensive immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Because of the sensor's high affinity, it primarily tracks rising concentrations of cytochrome C, capturing dynamic changes during apoptosis. This approach could help develop more advanced cancer disease models and apply to other complex in vitro disease models, such as organs-on-chips and organoids.

Purpose: This study investigated the potential of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 VD 3 ) to mitigate bisphenol A (BPA)-induced apoptosis in human ovarian granulosa KGN cells with the aim of establishing a theoretical foundation for understanding of how vitamin D improved ovarian function in patients with polycystic ovary syndrome (PCOS)., Methods: The impact of varying concentrations of BPA and 1,25(OH) 2 VD 3 on KGN cell viability was elucidated. It was established that BPA-induced apoptosis in KGN cells. Subsequently, KGN cells underwent pretreatment with 1,25(OH) 2 VD 3 , followed by exposure to BPA. The apoptosis rate, reactive oxygen species (ROS) levels, and mitochondrial function of the cells were meticulously assessed, along with the expression levels of genes associated with apoptosis as well as antioxidant and mitochondrial biogenesis., Results: BPA induced a notable increase in apoptosis (P < 0.001) and oxidative stress (P < 0.001) in KGN cells, accompanied by a significant reduction in mitochondrial membrane potential (P < 0.001) and severe impairment of mitochondrial function. Following pretreatment of KGN cells with 1,25(OH) 2 VD 3 , there was a significant decrease in the apoptosis rate (P = 0.004), coupled with a reduction in ROS production (P = 0.002). Concomitantly, the upregulation of PGC-1α (P = 0.009) and SOD (P = 0.018) was observed, while mRNA expression of BAX (P = 0.011), Cyt c (P = 0.001), Apaf-1 (P = 0.012), caspase-9 (P < 0.001), and caspase-3 (P = 0.011) was downregulated. Notably, the mitigation of mitochondrial damage was evident through restored mitochondrial membrane potential (P < 0.001), as corroborated by electron microscope results., Conclusions: 1,25(OH) 2 VD 3 mitigated BPA-induced damage and apoptosis in KGN cells by upregulating the expression of PGC-1α and impeding the mitochondrial cytochrome c (Cyt c) apoptotic pathway. This study established a novel theoretical foundation for utilizing vitamin D in the treatment of PCOS patients., (© 2024. The Author(s).)

Authentication of true (genuine) cow leathers is in high demand to promote merchandise and economic growth. The present study employs RT-PCR-based TaqMan assay to facilitate the identification. Species-specific primers and probes were designed utilizing the existing NCBI data on mitochondrial DNA (mtDNA) genes, particularly the cytochrome b region (Cyt b). Mitochondrial DNA extracted from leather samples of both Bos taurus and Bos indicus and analyzed following the appropriate procedures. The RT-PCR results showed the designed primers and probes are exceptionally precise for cow leather samples. The established detection limit for the assay is estimated as 0.1 ng of DNA. In summary, the amplifiable mtDNA extracted from finished leather enables the identification of authentic cow leathers using the RT-PCR TaqMan assay, representing a pioneering report in this field., 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 evolutionary dynamics of the ecoregions of southern South America and the species that inhabit them have been poorly studied, and few biogeographic hypotheses have been proposed and tested. Quaternary climatic oscillations are among the most important processes that have led to the current distribution of genetic variation in different regions of the world. In this work, we studied the evolutionary history and distribution of the Córdoba vesper mouse (Calomys venustus), a characteristic rodent of the region of which little is known about its natural history. Since the population dynamics of this species are influenced by climatic factors, this rodent is a suitable model to study the effects of Quaternary climatic oscillations in central Argentina. The mitochondrial cytochrome b gene was sequenced to analyze the phylogeography of C. venustus, and ecological niche modeling tools were used to map its potential distributions. The results of these approaches were combined to provide additional spatially explicit information about this species' past. Our results suggest that the Espinal was the area of origin of this species, which expanded demographically and spatially during the last glacial period. A close relationship was found between the Espinal and the Mountain Chaco. These results are consistent with previous studies and emphasize the role of the Espinal in the biogeographic history of southern South America as an area of origin of several species., (© 2024 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Purpose: We aimed to investigate the urinary caspase-3 and cytochrome c levels in patients with unilateral antenatal hydronephrosis and to determine whether changes in urinary biomarker levels could be useful for both predicting the need for surgical intervention due to ureteropelvic junction obstruction (UPJO) and postoperative surgical success., Methods: Sixty-five children with a history of unilateral antenatal hydronephrosis and postnatal anteroposterior diameter ≥ 10 mm were included in this prospective case-control study between January 2013 and December 2021. The obstruction group consisted of 33 patients (28 boys, 84.8%) who underwent open dismembered pyeloplasty due to UPJO. The non-obstructive dilatation (NOD) group consisted of 32 patients (27 boys, 84.4%) with stable or improving hydronephrosis and no significant reduction in ipsilateral split renal function during follow-up, whereas 34 healthy children were enrolled in the study as a control group. Urinary urinary caspase-3 and cytochrome c levels using ELISA were measured., Results: The median preoperative urinary caspase-3 level was significantly higher in the obstruction group when compared to the NOD group (4.82 ng/mgCr vs. 2.61 ng/mgCr, p = 0.013) as well as the control group (4.82 ng/mgCr vs. 1.72 ng/mgCr, p = 0.002). In the postoperative period, urinary caspase-3 levels significantly decreased compared to preoperative measurements (4.82 ng/mgCr vs. 2.51 ng/mgCr, p = 0.006) and became similar to the control group (2.51 ng/mgCr vs. 1.72 ng/mgCr, p = 0.422). On the other hand, no significant differences were observed in urinary cytochrome c levels between the groups. All patients who underwent pyeloplasty achieved postoperative resolution in hydronephrosis and improved drainage on MAG-3, so none of the patients required re-do pyeloplasty. Postoperative decrease in caspase-3 level was found to be compatible with adequate urine drainage on MAG-3 scan. The cut-off value of urinary caspase-3 to predict patients requiring pyeloplasty was found to be 3.31 ng/mg creatinine with 63.6% sensitivity, 62.5% specificity (AUC = 0.679). In the multivariable analysis, urinary caspase-3 level (OR: 1.653, p = 0.019), anteroposterior pelvic diameter (OR: 1.401, p = 0.001), and split renal function on MAG-3 (OR: 1.277, p = 0.011) were found to be independent factors in determining patients who require surgery., Conclusion: Based on our preliminary findings, urinary caspase-3 levels could be a useful biomarker not only for predicting the need for surgical intervention but also for determining the postoperative surgical success in children with UPJO., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Free interconversion of cytochrome C (CytC) between native ferrous (Cyt-Fe II ) and oxidized ferric (CytC-Fe III ) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC-Fe III to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt-Fe II to CytC-Fe III within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear-cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label-free detection of ferrous and ferric heme proteins., (© 2024 Federation of European Biochemical Societies.)

Background: Babesia spp. are protozoan parasites that infect the red blood cells of domesticated animals, wildlife and humans. A few cases of giant pandas (a flagship species in terms of wildlife conservation) infected with a putative novel Babesia sp. have been reported. However, comprehensive research on the morphological and molecular taxonomic classification of this novel Babesia sp. is still lacking. This study was designed to close this gap and formally describe this new Babesia sp. infecting giant pandas., Methods: Detailed morphological, molecular and phylogenetic analyses were conducted to characterise this Babesia sp. and to assess its systematic relationships with other Babesia spp. Blood samples from giant pandas infected with Babesia were subjected to microscopic examination. The 18S ribosomal RNA (18S rRNA), cytochrome b (cytb) and mitochondrial genome (mitogenome) of the new Babesia sp. were amplified, sequenced and assembled using DNA purified from blood samples taken from infected giant pandas. Based on the newly generated 18S rRNA, cytb and mitogenome sequences, phylogenetic trees were constructed., Results: Morphologically, the Babesia sp. from giant pandas exhibited various forms, including round to oval ring-shaped morphologies, resembling those found in other small canine Babesia spp. and displaying typical tetrads. Phylogenetic analyses with the 18S rRNA, cytb and mitogenome sequences revealed that the new Babesia sp. forms a monophyletic group, with a close phylogenetic relationship with the Babesia spp. that infect bears (Ursidae), raccoons (Procyonidae) and canids (Canidae). Notably, the mitogenome structure consisted of six ribosomal large subunit-coding genes (LSU1-6) and three protein-coding genes (cytb, cox3 and cox1) arranged linearly., Conclusions: Based on coupled morphological and genetic analyses, we describe a novel species of the genus Babesia, namely, Babesia ailuropodae n. sp., which infects giant pandas., (© 2024. The Author(s).)

Despite being endemic in Iraq, no reports have been published in the past 10 years to update the molecular epidemiology of the Old World screwworm fly (OWSF), Chrysomya bezziana, in this country. In the present study, 130 sheep from 10 Iraqi governorates were found infected with C. bezziana larvae, whose identities were PCR-confirmed based on the cytochrome b (Cytb) gene, and 23 isolates from various tested governorates were successfully sequenced. Although most isolates (n = 20) belonged to the common haplotype circulating in Iraq, two new haplotypes were detected. Significant changes in OWSF epidemiology in Iraq were also suggested, since infestations were detected, for the first time, in Nineveh governorate. Isolates of the present study were combined to those previously published from Iraq and worldwide, collected after searching the GenBank, and various genetic and population structure analyses were conducted. These isolates displayed a great statistically significant value when tested for the purifying (negative) selection, suggesting the limited occurrence of genetic variations, which was evidenced by the high sequence conservation (C = 0.937) value detected. A few isolates from Africa were revealed during our search, and clustered in a separate lineage other than that of the Asian isolates. The latter displayed different genetic variation patterns when compared. For example, isolates from geographically separate regions, e.g., the Gulf Arab countries and South-Eastern Asia had marked genetic differences. On the other hand, isolates from regions with close geographic proximity (the Gulf Arab countries and Iran) had limited genetic subdivision. This is not the case when comparing isolates from 10 islands in the Indonesian Archipelago. Populations from Sumatra and Sumba were isolated and displayed high genetic variations toward the other populations. On the contrary, populations from Sulawesi, Lombok and Sumbawa displayed limited genetic variations. This is particularly important, since it can help detecting the dynamics of establishing the sterile insect technique over various regions as an effective control strategy against the OWSFs., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Three nominal species of the killifish genus Aplocheilus are reported from the lowlands of Sri Lanka. Two of these, Aplocheilus dayi and Aplocheilus werneri, are considered endemic to the island, whereas Aplocheilus parvus is reported from both Sri Lanka and Peninsular India. Here, based on a collection from 28 locations in Sri Lanka, also including a dataset of Asian Aplocheilus downloaded from GenBank, we present a phylogeny constructed from the mitochondrial cytochrome b (cytb), mitochondrial cytochrome c oxidase subunit 1 (cox1), and nuclear recombination activating protein 1 (rag1), and investigate the interrelationships of the species of Aplocheilus in Sri Lanka. The endemic Sri Lankan aplocheilid clade comprising A. dayi and A. werneri is recovered as the sister group to the clade comprising A. parvus from Sri Lanka and Aplocheilus blockii from Peninsular India. The reciprocal monophyly of A. dayi and A. werneri is not supported in our molecular phylogeny. A. dayi and A. werneri display strong sexual dimorphism, but species-level differences are subtle, explained mostly by pigmentation patterns. Their phenotypes exhibit a parapatric distribution and may represent locally adapted forms of a single species. Alternatively, the present study does not rule out the possibility that A. dayi and A. werneri may represent an incipient species pair or that they have undergone introgression or hybridization in their contact zones. We provide evidence that the Nilwala-Gin region of southwestern Sri Lanka may have acted as a drought refugium for these fishes., (© 2024 Fisheries Society of the British Isles.)

Thrombocytopenia 4 (THC4) is an autosomal-dominant thrombocytopenia caused by mutations in CYCS, the gene encoding cytochrome c (CYCS), a small haeme protein essential for electron transport in mitochondria and cell apoptosis. THC4 is considered an extremely rare condition since only a few patients have been reported so far. These subjects presented mild thrombocytopenia and no or mild bleeding tendency. In this study, we describe six Italian families with five different heterozygous missense CYCS variants: p.Gly42Ser and p.Tyr49His previously associated with THC4, and three novel variants (p.Ala52Thr, p.Arg92Gly, and p.Leu99Val), which have been classified as pathogenic by bioinformatics and segregation analyses. Moreover, we supported functional effects of p.Ala52Thr and p.Arg92Gly on oxidative growth and respiratory activity in a yeast model. The clinical characterization of the 22 affected individuals, the largest series of THC4 patients ever reported, showed that this disorder is characterized by mild-to-moderate thrombocytopenia, normal platelet size, and function, low risk of bleeding, and no additional clinical phenotypes associated with reduced platelet count. Finally, we describe a significant correlation between the region of CYCS affected by mutations and the extent of thrombocytopenia, which could reflect different degrees of impairment of CYCS functions caused by different pathogenetic variants., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Background: The two-spotted spider mite Tetranychus urticae causes significant damage to ornamental, cotton, sugarcane and horticultural crops in Australia. It has a long history of developing resistance to many acaricides including bifenazate. A mutation in the conserved cd1- and ef-helices of the Qo pocket of cytochrome b is recognized as the primary mechanism of bifenazate resistance. To investigate the resistance mechanisms against bifenazate in Australian two-spotted spider mite, we sequenced the complete mitochondrion genome of five mite strains including a susceptible and bifenazate-resistant strain., Results: We identified a novel mutation D252N in the G126S background at cytochrome b being the cause of bifenazate resistance in a bifenazate-resistant strain, Bram. We validated the role of this mutation combination by reciprocal crosses between a bifenazate resistant and susceptible strain. By doing these crosses we confirmed the pattern of inheritance was maternal. Additionally, mitochondrial heteroplasmy was not observed by single mite genotyping of the mutations in cytb in a known bifenazate-resistant strain Bram. The phylogenetic analysis with the complete mitochondrion genome sequences revealed that Australian two-spotted spider mite strains are closely related to the green form of T. urticae found in China., Conclusions: The novel mutation D252N found in the cytochrome b in the G126S background was revealed to be the main cause of bifenazate resistance in the Australian T. urticae strain Bram. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c -type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast iso -1 cytochrome c to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins.

Montmorillonite (MM) crystal nanoplates acquire anticancer properties when coated with the mitochondrial protein cytochrome c (cytC) due to the cancer cells' capability to phagocytize cytC-MM colloid particles. The introduced exogenous cytC initiates apoptosis: an irreversible cascade of biochemical reactions leading to cell death. In the present research, we investigate the organization of the cytC layer on the MM surface by employing physicochemical and computer methods-microelectrophoresis, static, and electric light scattering-to study cytC adsorption on the MM surface, and protein electrostatics and docking to calculate the local electric potential and Gibbs free energy of interacting protein globules. The found protein concentration dependence of the adsorbed cytC quantity is nonlinear, manifesting a positive cooperative effect that emerges when the adsorbed cytC globules occupy more than one-third of the MM surface. Computer analysis reveals that the cooperative effect is caused by the formation of protein associates in which the cytC globules are oriented with oppositely charged surfaces. The formation of dimers and trimers is accompanied by a strong reduction in the electrostatic component of the Gibbs free energy of protein association, while the van der Waals component plays a secondary role.

The human steroidogenic cytochrome P450 CYP17A1 catalyzes two types of reactions in the biosynthetic pathway leading from pregnenolone to testosterone and several other steroid hormones. The first is the hydroxylation of pregnenolone or progesterone to the corresponding 17α-hydroxy steroid, followed by a lyase reaction that converts these 17α-hydroxy intermediates to the androgens dehydroepiandrosterone and androstenedione, respectively. cytochrome b 5 (cyt b 5 ) is known to act as both an effector and electron donor for the lyase oxidations, markedly stimulating the rate of the lyase reaction in its presence relative to the rate in its absence. Extensive sequential backbone 1 H, 15 N and 13 C nuclear magnetic resonance assignments have now been made for oxidized CYP17A1 bound to the prostate cancer drug and inhibitor abiraterone. This is the first eukaryotic P450 for which such assignments are now available. These assignments allow more complete interpretation of the structural perturbations observed upon cyt b 5 addition. Possible mechanism(s) for the effector activity of cyt b 5 are discussed in light of this new information.

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.)