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The long acclimation period and sensitivity to environmental conditions of Anammox are the bottlenecks for its promotion and application. An innovative strategy was adopted to accelerate functional microbial enhancement and improve nitrogen removal performance by inoculating cryopreserved Anammox sludge and activated sludge with intermittent dosing of nanoscale zero-valent iron (nZVI). The acclimation time was shortened by 76 days with nitrogen removal efficiency (NRE) reaching up to 91.07 %. Anammox, NDFO (nitrate/nitrite-dependent Fe(II) oxidation), Feammox (Fe(III) reduction coupled with anaerobic ammonium oxidation) and abiotic reactions were coupled in the system with nZVI, contributing to 69.79 %, 15.14 %, 9.84 % and 0.25 % of nitrogen removal, respectively. Further microbial analysis demonstrated significant enrichment of functional microorganisms, such as Candidatus Jettenia, Acidovorax and Comamonas. High-efficient nitrogen removal was attribute to the increase of functional genes involved in Anammox, electronic transfer, heme C synthesis and iron metabolism. This work provides an inspiring idea for the mainstream Anammox application., 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.)

The present work is another part of our investigation on the pathway of dissimilatory sulfate reduction and covers a theoretical study on the reaction catalyzed by dissimilatory sulfite reductase (dSIR). dSIR is the terminal enzyme involved in this metabolic pathway, which uses the siroheme-[4Fe4S] cofactor for six-electron reduction of sulfite to sulfide. In this study we use a large cluster model containing siroheme-[4Fe4S] cofactor and protein residues involved in the direct interactions with the substrate, to get insight into the most feasible reaction mechanism and to understand the role of each considered active site component. In combination with earlier studies reported in the literature, our results lead to several interesting insights. One of the most important conclusions is that the reaction mechanism consists of three steps of two-electron reduction of sulfur and the probable role of the siroheme-[4Fe4S] cofactor is to ensure the delivery of packages of two electrons to the reactant., (© 2024 Wiley-VCH GmbH.)

Artificial syntheses of biologically active molecules have been fruitful in many bioinspired catalysis applications. Specifically, verdoheme and biliverdin, bearing polypyrrole frameworks, have inspired catalyst designs to address energy and environmental challenges. Despite remarkable progress in benchtop synthesis of verdoheme and biliverdin derivatives, all reported syntheses, starting from metalloporphyrins or inaccessible biliverdin precursors, require multiple steps to achieve the final desired products. Additionally, such synthetic procedures use multiple reactants/redox agents and involve multistep purification/extraction processes that often lower the yield. However, in a single step using atmospheric oxygen, heme oxygenases selectively generate verdoheme or biliverdin from heme. Motivated by such enzymatic pathways, we report a single-step electrosynthesis of verdoheme or biliverdin derivatives from their corresponding meso-aryl-substituted metalloporphyrin precursors. Our electrosynthetic methods have produced a copper-coordinating verdoheme analog in >80% yield at an applied potential of 0.65 V vs ferrocene/ferrocenium in air-exposed acetonitrile solution with a suitable electrolyte. These electrosynthetic routes reached a maximum product yield within 8 h of electrolysis at room temperature. The major products of verdoheme and biliverdin derivatives were isolated, purified, and characterized using electrospray mass spectrometry, absorption spectroscopy, cyclic voltammetry, and nuclear magnetic resonance spectroscopy techniques. Furthermore, X-ray crystallographic data were collected for select cobalt (Co)- and Cu-chelating verdoheme and metal-free biliverdin products. Electrosynthesis routes for the selective modification at the macrocycle ring in a single step are not known yet, and therefore, we believe that this report would advance the scopes of electrosynthesis strategies.

Insulin deficiency in type 1 diabetes (T1D) leads to an impairment of glucose metabolism and mitochondrial function. Actovegin is a hemodialysate of calf blood, which has been shown to enhance glucose uptake and cell metabolism in healthy human skeletal muscle. The objectives of this study were to determine the effects of Actovegin on skeletal muscle mitochondrial respiration and functional aerobic capacity in a T1D mouse model. Effects on the expression of mitochondrial proteins, body mass, and food and water consumption were also investigated. Streptozotocin-induced T1D male C57B1/6 mice (aged 3-4 months) were randomized to an Actovegin group and a control group. Every third day, the Actovegin and control groups were injected intraperitoneally with (0.1 mL) Actovegin and (0.1 mL) physiological salt solution, respectively. Oxidative phosphorylation (OXPHOS) capacity of the vastus lateralis muscle was measured by high resolution respirometry in addition to the expression levels of the mitochondrial complexes as well as voltage-dependent anion channel. Functional aerobic capacity was measured using a rodent treadmill protocol. Body mass and food and water consumption were also measured. After 13 days, in comparison to the control group, the Actovegin group demonstrated a significantly higher skeletal muscle mitochondrial respiratory capacity in an ADP-restricted and ADP-stimulated environment. The Actovegin group displayed a significantly lesser decline in functional aerobic capacity and baseline body mass after 13 days. There were no significant differences in food or water consumption between groups. Actovegin could act as an effective agent for facilitating glucose metabolism and improving OXPHOS capacity and functional aerobic capacity in T1D. Further investigation is warranted to establish Actovegin's potential as an alternative therapeutic drug for T1D., Competing Interests: The authors declare there are no competing interests.

Background: Actovegin is a hemodialysate of calf's blood and has been used for several decades in the countries of Central Asia, East Asia, Russia and some European countries. It has been used to treat patients with various neurological conditions, vascular disorders, and ischemic stroke., Objectives: To perform a systematic review to evaluate the effect of Actovegin in patients who have suffered an ischemic stroke., Methods: A search of MEDLINE, PubMed, Cochrane and Embase was carried out from inception to October 10, 2021 for clinical trials and observational studies with a control group, published in English or Russian., Results: Of 220 identified unique records, 84 full-text articles were screened, and 5 studies were selected that met the inclusion criteria. This included 4 observational studies with control groups and one randomized, placebo-controlled clinical trial. These studies enrolled a total of 3879 patients of which 720 patients received Actovegin administered intravenously and/or orally for a duration ranging from 10 to 180 days. Because of study heterogeneity, meta-analysis was not performed. No consistent evidence on improved survival, quality of life, neurologic symptoms, activities of daily living or disability was identified. One study showed statistically significant improvements in the Alzheimer's Disease Assessment Scale, cognitive subscale, extended version (ADAS-cog+) for Actovegin compared with placebo at 6 months but the clinical relevance of this change is uncertain. One study reported a higher incidence of recurrent ischemic stroke, transient ischemic attack or intracerebral hemorrhage in patients taking Actovegin compared to placebo., Conclusions: The benefits of Actovegin are uncertain and that there is potential risk of harm in patients with stroke. More evidence is needed from rigorously designed clinical trials to justify the role of Actovegin in patients with ischemic stroke., Competing Interests: The authors have declared that no competing interests exist.

The branched aerobic respiratory chain in Bacillus cereus comprises three terminal oxidases: cytochromes aa3 , caa3 , and bd . Cytochrome caa3 requires heme A for activity, which is produced from heme O by heme A synthase (CtaA). In this study, we deleted the ctaA gene in B. cereus AH187 strain, this deletion resulted in loss of cytochrome caa3 activity. Proteomics data indicated that B. cereus grown in glucose-containing medium compensates for the loss of cytochrome caa3 activity by remodeling its respiratory metabolism. This remodeling involves up-regulation of cytochrome aa3 and several proteins involved in redox stress response-to circumvent sub-optimal respiratory metabolism. CtaA deletion changed the surface-composition of B. cereus, affecting its motility, autoaggregation phenotype, and the kinetics of biofilm formation. Strikingly, proteome remodeling made the ctaA mutant more resistant to cold and exogenous oxidative stresses compared to its parent strain. Consequently, we hypothesized that ctaA inactivation could improve B. cereus fitness in a nutrient-limited environment.

Aerobic respiration is a key energy-producing pathway in many prokaryotes and virtually all eukaryotes. The final step of aerobic respiration is most commonly catalyzed by heme-copper oxidases embedded in the cytoplasmic or mitochondrial membrane. The majority of these terminal oxidases contain a prenylated heme (typically heme a or occasionally heme o ) in the active site. In addition, many heme-copper oxidases, including mitochondrial cytochrome c oxidases, possess a second heme a cofactor. Despite the critical role of heme a in the electron transport chain, the details of the mechanism by which heme b , the prototypical cellular heme, is converted to heme o and then to heme a remain poorly understood. Recent structural investigations, however, have helped clarify some elements of heme a biosynthesis. In this review, we discuss the insight gained from these advances. In particular, we present a new structural model of heme o synthase (HOS) based on distance restraints from inferred coevolutionary relationships and refined by molecular dynamics simulations that are in good agreement with the experimentally determined structures of HOS homologs. We also analyze the two structures of heme a synthase (HAS) that have recently been solved by other groups. For both HOS and HAS, we discuss the proposed catalytic mechanisms and highlight how new insights into the heme-binding site locations shed light on previously obtained biochemical data. Finally, we explore the implications of the new structural data in the broader context of heme trafficking in the heme a biosynthetic pathway and heme-copper oxidase assembly.

In this study on model compounds for the resting oxidized state of the iron‑copper binuclear center in cytochrome c oxidase (CcO), we describe the synthesis of a new μ-oxo-heme/Cu complex, [(TPP)Fe III -O-Cu II (tmpa)][B(C 6 F 5 ) 4 ] (2) {TPP: tetraphenyl porphyrinate(2-); TMPA: tris(2-pyridylmethylamine)}, as well as two protonation events for three μ-oxo-heme/Cu complexes with varying peripheral substituents on the heme site. The addition of increasing amounts of strong acid to these μ-oxo-heme/Cu systems successively led to the generation of the corresponding μ-hydroxo, μ-aquo, and the dissociated complexes. The heme/Cu assemblies bridged through a water ligand are reported here for the first time and the 1 H NMR and 19 F NMR spectral properties are consistent with antiferromagnetically coupled high-spin iron(III) and copper(II) centers. By titration using a series of protonated amines, the pK a values for the corresponding μ-hydroxo-heme/Cu species (i.e., the first protonation event) have been reported and compared with the pK a ranges previously estimated for related systems. These synthetic systems may represent structural models for the oxidized Fe III -X-Cu II resting state, or turnover intermediates and can be employed to clarify the nature of proton/electron transfer events in CcO. SYNOPSIS: The resting oxidized state of the cytochrome c oxidase active site contains an Fe a3 -OH x -Cu B moiety. Here, we investigated two successive protonation events, for a series of μ-oxo-heme/Cu assemblies and reported the pK a values for the first protonation event. The μ-aquo-heme/Cu complexes described here are the first examples of such systems., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cytochrome c oxidase (C c O) pumps protons from the N-side to the P-side and consumes electrons from the P-side of the mitochondrial membrane driven by energy gained from reduction of dioxygen to water. ATP synthesis uses the resulting proton gradient and electrostatic potential difference. Since the distance a proton travels through C c O is too large for a one-step transfer process, proton-loading sites (PLS) that can carry protons transiently are necessary. One specific pump-active PLS couples to the redox reaction, thus energizing the proton to move across the membrane against electric potential and proton gradient. The PLS should also prevent proton backflow. Therefore, the propionates of the two redox-active hemes in C c O were suggested as PLS candidates although, according to C c O crystal structures, none of the four propionates can be protonated on account of strong H-bonds. Here, we show that modeling the local structure around heme a 3 propionates enhances significantly their capability of carrying a proton jointly. This was not possible for the propionates of heme a . The modeled structures are stable in molecular dynamics simulations (MDS) and are energetically similar to the crystal structure. Precise electrostatic energy computations of MDS data are used to estimate the p K A values of all titratable residues in C c O. For the modeled structures, the heme a 3 propionates have p K A values high enough to host a proton transiently but not too high to fix the proton permanently. The change in p K A throughout the redox reaction is sufficient to push the proton to the P-side of the membrane and to provide the protons with the necessary amount of energy for ATP synthesis.

Heme enzymes are some of the most versatile catalysts in nature. In recent years it has been found that they can also catalyze reactions for which there are no equivalents in nature. This development has been driven by the abiological catalytic reactivity reported for bio-inspired and biomimetic iron porphyrin complexes. This review focuss es on heme enzymes for catalysis of cyclopropanation reactions. The two most important approaches used to create enzymes for cyclopropanation are repurposing of heme enzymes and the various strategies used to improve these enzymes such as mutagenesis and heme replacement, and artificial heme enzymes. These strategies are introduced and compared. Moreover, lessons learned with regard to mechanism and design principles are discussed., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Archaeal methanogens, methanotrophs, and alkanotrophs have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, traffic, deploy, and store these elements. Here, we examined the distribution of homologs of proteins mediating key steps in Fe/S metabolism in model microorganisms, including iron(II) sensing/uptake (FeoAB), sulfide extraction from cysteine (SufS), and the biosynthesis of iron-sulfur [Fe-S] clusters (SufBCDE), siroheme (Pch2 dehydrogenase), protoheme (AhbABCD), cytochrome c (Cyt c ) (CcmCF), and iron storage/detoxification (Bfr, FtrA, and IssA), among 326 publicly available, complete or metagenome-assembled genomes of archaeal methanogens/methanotrophs/alkanotrophs. The results indicate several prevalent but nonuniversal features, including FeoB, SufBC, and the biosynthetic apparatus for the basic tetrapyrrole scaffold, as well as its siroheme (and F 430 ) derivatives. However, several early-diverging genomes lacked SufS and pathways to synthesize and deploy heme. Genomes encoding complete versus incomplete heme biosynthetic pathways exhibited equivalent prevalences of [Fe-S] cluster binding proteins, suggesting an expansion of catalytic capabilities rather than substitution of heme for [Fe-S] in the former group. Several strains with heme binding proteins lacked heme biosynthesis capabilities, while other strains with siroheme biosynthesis capability lacked homologs of known siroheme binding proteins, indicating heme auxotrophy and unknown siroheme biochemistry, respectively. While ferritin proteins involved in ferric oxide storage were widespread, those involved in storing Fe as thioferrate were unevenly distributed. Collectively, the results suggest that differences in the mechanisms of Fe and S acquisition, deployment, and storage have accompanied the diversification of methanogens/methanotrophs/alkanotrophs, possibly in response to differential availability of these elements as these organisms evolved. IMPORTANCE Archaeal methanogens, methanotrophs, and alkanotrophs, argued to be among the most ancient forms of life, have a high demand for iron (Fe) and sulfur (S) for cofactor biosynthesis, among other uses. Here, using comparative bioinformatic approaches applied to 326 genomes, we show that major differences in Fe/S acquisition, trafficking, deployment, and storage exist in this group. Variation in these characters was generally congruent with the phylogenetic placement of these genomes, indicating that variation in Fe/S usage and deployment has contributed to the diversification and ecology of these organisms. However, incongruency was observed among the distribution of cofactor biosynthesis pathways and known protein destinations for those cofactors, suggesting auxotrophy or yet-to-be-discovered pathways for cofactor biosynthesis.

Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H 2 O 2 to water and oxygen, but it is also able to oxidase various small organic compounds including catechol and phenol. The crystal structure of Scytalidium catalase reveals the presence of three linked channels providing access to the exterior like other catalases reported so far. The function of these channels has been extensively studied, revealing the possible routes for substrate flow and product release. In this report, we have focussed on the semi-conserved residue Val228, located near to the vinyl groups of the heme at the opening of the lateral channel. Its replacement with Ala, Ser, Gly, Cys, Phe and Ile were tested. We observed a significant decrease in catalytic efficiency in all mutants with the exception of a remarkable increase in oxidase activity when Val228 was mutated to either Ala, Gly or Ser. The reduced catalytic efficiencies are characterized in terms of the restriction of hydrogen peroxide as electron acceptor in the active centre resulting from the opening of lateral channel inlet by introducing the smaller side chain residues. On the other hand, the increased oxidase activity is explained by allowing the suitable electron donor to approach more closely to the heme. The crystal structures of V228C and V228I were determined at 1.41 and 1.47 Å resolution, respectively. The lateral channels of the V228C and V228I presented a broadly identical chain of arranged waters to that observed for wild-type enzyme., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cytochrome c oxidase in the respiratory chain of bacteria and mitochondria couples the reduction of molecular oxygen to form water with the generation of a transmembrane proton gradient. Bacillus subtilis has two heme A-containing heme-copper oxidases: the menaquinol oxidase cytochrome aa 3 and the cytochrome c oxidase cytochrome caa 3 . By screening three collections of mutants for defective cytochrome c oxidase, we found the genes for two, new membrane-bound assembly factors in B. subtilis: ytkA and yozB (renamed ctaK and ctaM, respectively). CtaK is a lipoprotein without sequence similarity to any protein of known function. We show that CtaK functions together with Sco1 (YpmQ) in a pathway, leading to the assembly of the Cu A center in cytochrome caa 3 and seems to be a functional analogue to proteins of the periplasmic Cu A chaperone family (PCu A C). CtaM is required for the activity of both cytochrome caa 3 and cytochrome aa 3 and dispensable for the insertion of heme A into these oxidases. The orthologous Bacillus anthracis protein and the distantly related Staphylococcus aureus CtaM complemented CtaM deficiency in B. subtilis, establishing a common function of CtaM in these bacteria. As the overall result of our work, 12 different proteins are known to function in the biosynthesis of cytochrome c oxidase in B. subtilis., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Formation of five coordinated ferric (ferrous) verdoheme oxygenase complexes have been investigated at ωB97X-D/6-31G(d) level of theory. This process was carried out by adsorption of imidazole and human/mouse verdoheme oxygenase (VO) compounds. Global reactivity indexes show electrophile and nucleophile roles of the VO complexes and Imidazole, respectively. This result confirms their interaction, molecular electrostatic potential (MEP) maps, and low HOMO FRVMO -LUMO Imidazole gap. These interactions can cause in adsorption and five coordinated of the VO complexes. More negative value (-64.3 kJ mol -1 ) of adsorption energy (E ads ) in the FRVMO complex shows better adsorption strength and stable configuration. Significant point of this interaction is hydrogen transfer from imidazole to the nearest oxygen of the VO complexes; this issue is approved using quantum theory of atom in molecule (QTAIM) and natural bond orbital (NBO) analysis. QTAIM calculations confirm ionic bonding between the transferred hydrogen and the oxygen atom of the VO. The 312.2-kcal mol -1  s order stabilization energies in this complex are confirmation for strong donation and better formation of five coordinated complex in electron view point.

Bacterial response to nitric oxide (NO) is of major importance for bacterial survival. NO stress is a main actor of the eukaryotic immune response and several pathogenic bacteria have developed means for detoxification and repair of the damages caused by NO. However, bacterial mechanisms of NO resistance by Gram-positive bacteria are poorly described. In the opportunistic foodborne pathogen Bacillus cereus , genome sequence analyses did not identify homologs to known NO reductases and transcriptional regulators, such as NsrR, which orchestrate the response to NO of other pathogenic or non-pathogenic bacteria. Using a transcriptomic approach, we investigated the adaptation of B. cereus to NO stress. A cluster of 6 genes was identified to be strongly up-regulated in the early phase of the response. This cluster contains an iron-sulfur cluster repair enzyme, a nitrite reductase and three enzymes involved in siroheme biosynthesis. The expression pattern and close genetic localization suggest a functional link between these genes, which may play a pivotal role in the resistance of B. cereus to NO stress during infection.

Photosynthetic electron transfers occur through multiple components ranging from small soluble proteins to large integral membrane protein complexes. Co-crystallization of a bacterial photosynthetic electron transfer complex that employs weak hydrophobic interactions was achieved by using high-molar-ratio mixtures of a soluble donor protein (high-potential iron-sulfur protein, HiPIP) with a membrane-embedded acceptor protein (reaction center, RC) at acidic pH. The structure of the co-complex offers a snapshot of a transient bioenergetic event and revealed a molecular basis for thermodynamically unfavorable interprotein electron tunneling. HiPIP binds to the surface of the tetraheme cytochrome subunit in the light-harvesting (LH1) complex-associated RC in close proximity to the low-potential heme-1 group. The binding interface between the two proteins is primarily formed by uncharged residues and is characterized by hydrophobic features. This co-crystal structure provides a model for the detailed study of long-range trans-protein electron tunneling pathways in biological systems.

PURPOSE： To explore the clinical effect of Actovegin in the treatment of acute oral mucositis in patients with radiotherapy. METHODS： One hundred and thirteen patients with acute oral mucositis caused by radiotherapy for nasopharyngeal carcinoma admitted to the Department of Oncology, the Fifth People's Hospital of Qinghai Province from July 2015 to September 2017 were randomly divided into the experimental group (57 cases) and control group (56 cases). Patients in the experimental group were treated with Aiweizhi, while patients in the control group were treated with new rehabilitation. The changes of VAS score, oral mucositis grade, serum CRP, IL-6, TGF-β1 and TNF-α were compared between the 2 groups. The data were analyzed with SPSS 16.0 software package. RESULTS: Before treatment, there was no significant difference in VAS score between the 2 groups (P>0.05). After 1 week and 2 weeks of treatment, the VAS scores of the two groups were significantly lower than those before treatment (P<0.05). The VAS score of the experimental group was significantly lower than that of the control group (P<0.05). After 2 weeks of treatment, oral mucositis grade of the experimental group was significantly lower than that of the control group (P<0.05). There was no significant difference in serum CRP, IL-6, TGF-β1, and TNF-α level between the 2 groups before treatment (P>0.05). After 2 weeks of treatment, the level of serum CRP, IL-6, TGF-β1, and TNF-α in both groups was significantly lower than that before treatment (P<0.05). The serum level of CRP, IL-6, TGF-β1 and TNF-α in the experimental group was significantly lower than that in the control group (P<0.05). CONCLUSIONS: Actovegin has a clear clinical effect on acute oral mucositis in patients with radiotherapy, which can significantly alleviate the pain of patients and reduce the level of serum inflammatory factors.

Objective: To compare the antioxidant effects of cortexin, cerebrolysin and actovegin in rats with chronic brain ischemia., Material and Methods: Chronic brain ischemia was modeled in male rats by 50% stenosis of the common carotid arteries. Forty days after surgery, the animals received 2 ten-day courses of therapy, separated by a break of 10 days. Placebo, cortexin (0.3, 1 and 3 mg/kg), cerebrolysin (0.8, 2.5 and 7.5 ml/kg) and actovegin (5 ml/kg) were administered to animals as treatment. The concentration of malondialdehyde (MDA) in the homogenates was determined by the reaction with thiobarbituric acid, the concentration of reduced glutathione was determined by the reduction reaction of 5.5-dithiobis- (2-nitrobenzoic acid); determination of catalase activity, as well as the content of lactate and pyruvate, by commercially available reagent kits. The activity of superoxide dismutase (SOD) was determined by the photometric method based on an assessment of the degree of inhibition of the epinephrine oxidation reaction. All reactions were carried out in triplicates., Results: Modeling of chronic brain ischemia led to the statistically significant decrease in the content of lactate and pyruvate ( p <0.001, when compared with the control group), which was not accompanied by a significant decrease in their ratio ( p >0.05), as well as to the decrease in SOD, catalase activity, restored glutathione and increase in MDA concentrations. Compared with the control group, in the groups that received cortexin at a dose of 3 mg/kg/day, cerebrolysin at a dose of 7.5 ml/kg/day and actovegin at a dose of 5 ml/kg/day, there were an increase in the content of lactate and pyruvate (without a significant change in their ratio), restoration of glutathione levels and the activity of SOD and, to a lesser extent, catalase, combined with a decrease in the concentration of MDA., Conclusion: Course administration of cortexin (3 mg/kg), cerebrolysin (7.5 ml/kg) and, to a lesser extent, actovegin (5 ml/kg) has a positive effect on the state of the antioxidant system of the brain in rats with chronic brain ischemia.

Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa can respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes, for example, to the formation of biofilm, hence increasing difficulties in eradicating P. aeruginosa infections. A central step in denitrification is the reduction of nitrite to nitric oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme d 1 . While heme d 1 biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydroheme d 1 , the last intermediate of heme d 1 biosynthesis. We found that NirF forms a bottom-to-bottom β-propeller homodimer and confirmed this by multi-angle light and small-angle X-ray scattering. The N termini are adjacent to each other and project away from the core structure, which hints at simultaneous membrane anchoring via both N termini. Further, the complex with dihydroheme d 1 allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a ΔnirF strain of P. aeruginosa. Together, these data suggest that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d 1 derivatives. DATABASE: Structural data are available in PDB database under the accession numbers 6TV2 and 6TV9., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Heme oxygenases (HOs) play a critical role in recouping iron from the labile heme pool. The acquisition and liberation of heme iron are especially important for the survival of pathogenic bacteria. All characterized HOs, including those belonging to the HugZ superfamily, preferentially cleave free b-type heme. Another common form of heme found in nature is c-type heme, which is covalently linked to proteinaceous cysteine residues. However, mechanisms for direct iron acquisition from the c-type heme pool are unknown. Here we identify a HugZ homolog from the oligopeptide permease (opp) gene cluster of Paracoccus denitrificans that lacks any observable reactivity with heme b and show that it instead rapidly degrades c-type hemopeptides. This c-type heme oxygenase catalyzes the oxidative cleavage of the model substrate microperoxidase-11 at the β- and/or δ-meso position(s), yielding the corresponding peptide-linked biliverdin, CO, and free iron. X-ray crystallographic analysis suggests that the switch in substrate specificity from b-to c-type heme involves loss of the N-terminal α/β domain and C-terminal loop containing the coordinating histidine residue characteristic of HugZ homologs, thereby accommodating a larger substrate that provides its own iron ligand. These structural features are also absent in certain heme utilization/storage proteins from human pathogens that exhibit low or no HO activity with free heme. This study thus expands the scope of known iron acquisition strategies to include direct oxidative cleavage of heme-containing proteolytic fragments of c-type cytochromes and helps to explain why certain oligopeptide permeases show specificity for the import of heme in addition to peptides., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ammonia-oxidizing bacteria (AOB) convert ammonia (NH 3 ) to nitrite (NO 2 - ) as their primary metabolism and thus provide a blueprint for the use of NH 3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH 2 OH) to NO 2 - . HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other c hemes in each monomer that mediate electron transfer. Heme P460 cofactors are c -heme-based cofactors that have atypical protein cross-links between the peptide backbone and the porphyrin macrocycle. This cofactor has been observed in both the HAO and cytochrome (cyt) P460 protein families. However, there are differences; specifically, HAO uses a single tyrosine residue to form two covalent attachments to the macrocycle whereas cyt P460 uses a lysine residue to form one. In Nitrosomonas europaea , which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NH 2 OH and were both originally reported to produce NO 2 - . Each can inspire means to effect controlled release of chemical energy.Spectroscopically studying the P460 cofactors of HAO is complicated by the 21 non-P460 heme cofactors, which obscure the active site. However, monoheme cyt P460 is more approachable biochemically and spectroscopically. Thus, we have used cyt P460 to study biological NH 2 OH oxidation. Under aerobic conditions substoichiometric production of NO 2 - was observed along with production of nitrous oxide (N 2 O). Under anaerobic conditions, however, N 2 O was the exclusive product of NH 2 OH oxidation. We have advanced our understanding of the mechanism of this enzyme and have showed that a key intermediate is a ferric nitrosyl that can dissociate the bound nitric oxide (NO) molecule and react with O 2 , thus producing NO 2 - abiotically. Because N 2 O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO 2 - is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO 2 - enzymatically, but unlike cyt P460, its final product is NO, establishing it as an intermediate of nitrification. More broadly, NO can be recognized as a molecule common to the primary metabolisms of all organisms involved in nitrogen "defixation".Delving deeper into cyt P460 yielded insights broadly applicable to controlled biochemical redox processes. Studies of an inactive cyt P460 from Nitrosomonas sp. AL212 showed that this enzyme was unable to oxidize NH 2 OH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active N. europaea cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature's key to controlled oxidation of NH 2 OH. A key lesson of bioinorganic chemistry is reinforced: the polypeptide matrix is an essential part of dictating function. Our work also exposed some key functional contributions of noncanonical heme-protein cross-links. The heme-Lys cross-link of cyt P460 enforces the relative position of the cofactor and second-sphere residues. Moreover, the cross-link prevents the dissociation of the axial histidine residue, which stops catalysis, emphasizing the importance of this unique post-translational modification.

Cytochrome bd-I is a terminal oxidase of the Escherichia coli respiratory chain. This integral membrane protein contains three redox-active prosthetic groups (hemes b 558 , b 595 , and d) and couples the electron transfer from quinol to molecular oxygen to the generation of proton motive force, as one of its important physiological functions. The study was aimed at examining the effect of the membrane environment on the ligand-binding properties of cytochrome bd-I by absorption spectroscopy. The membrane environment was found to modulate the ligand-binding characteristics of the hemoprotein in both oxidized and reduced states. Absorption changes upon the addition of exogenous ligands, such as cyanide or carbon monoxide (CO), to the detergent-solubilized enzyme were much more significant and heterogeneous than those observed with the membrane-bound enzyme. In the native membranes, both cyanide and CO interacted mainly with heme d. An additional ligand-binding site (heme b 558 ) appeared in the isolated enzyme, as was evidenced by more pronounced changes in the absorption in the Soret band. This additional reactivity could also be detected after treatment of E. coli membranes with a detergent. The observed effect did not result from the enzyme denaturation, since reconstitution of the isolated enzyme into azolectin liposomes restored the ligand-binding pattern close to that observed for the intact membranes.

Purpose: The effect of Actovegin® was investigated on PMA- and LPS-induced human peripheral blood mononuclear cells (PBMCs)., Methods: PBMCs (1 × 10 6 cells/ml) from five blood donors (2 f, 3 m; 45-55 years) were grown in medium and exposed to Actovegin® in the presence or absence of PMA or LPS. Supernatants were collected to assess the concentration of cytokines (TNF-α, IL-1beta, IL-6 and IL-10). The reactive oxygen species (ROS) were assessed by a ROS-Glo TM H 2 O 2 assay., Results: Stimulation of cells by PMA or LPS (without Actovegin®) significantly increased the secretion of IL-1beta, IL-6, IL-10 and TNF-α from PBMCs, compared to controls. Pre-treatment of cells with Actovegin® (1, 5, 25, 125 µg/ml) plus PMA significantly decreased the secretion of IL-1beta from PBMCs, compared to controls (PMA without Actovegin®). In contrast, addition of Actovegin® (1, 5, 25, 125 and 250 µg/ml) plus LPS did not alter the IL-1beta production, compared to controls (LPS without Actovegin®). TNF-α, IL-6 and IL-10 do not contribute to the reduction of inflammatory reactions with Actovegin®., Conclusions: Actovegin® can reduce the PMA-induced IL-1beta release and the ROS production from PBMCs. These findings may help to explain the clinically known positive effects of Actovegin® on athletic injuries with inflammatory responses (e.g., muscle injuries, tendinopathies).

The integral membrane protein heme A synthase (HAS) catalyzes the biosynthesis of heme A, which is a prerequisite for cellular respiration in a wide range of aerobic organisms. Previous studies have revealed that HAS can form homo-oligomeric complexes, and this oligomerization appears to be evolutionarily conserved among prokaryotes and eukaryotes and is shown to be essential for the biological function of eukaryotic HAS. Despite its importance, little is known about the detailed structural properties of HAS oligomers. Here, we aimed to address this critical issue by analyzing the oligomeric state of HAS from Aquifex aeolicus (AaHAS) using a combination of techniques, including size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), cross-linking, laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS), and single-particle electron cryomicroscopy (cryo-EM). Our results show that HAS forms a thermostable trimeric complex. A cryo-EM density map provides information on the oligomerization interface of the AaHAS trimer. These results provide structural insights into HAS multimerization and expand our knowledge of this important enzyme. IMPORTANCE Heme A is a vital redox cofactor unique for the terminal cytochrome c oxidase in mitochondria and many microorganisms. It plays a key role in oxygen reduction by serving as an electron carrier and as the oxygen-binding site. Heme A is synthesized from heme O by an integral membrane protein, heme A synthase (HAS). Defects in HAS impair cellular respiration and have been linked to various human diseases, e.g., fatal infantile hypertrophic cardiomyopathy and Leigh syndrome. HAS exists as a stable oligomeric complex, and studies have shown that oligomerization of eukaryotic HAS is necessary for its proper function. However, the molecular architecture of the HAS oligomeric complex has remained uncharacterized. The present study shows that HAS forms trimers and reveals how the oligomeric arrangement contributes to the complex stability and flexibility, enabling HAS to perform its catalytic function effectively. This work provides the basic understanding for future studies on heme A biosynthesis., (Copyright © 2020 Zeng et al.)

Responses of a microbial community in the completely autotrophic nitrogen removal over nitrite (CANON) process, which was shocked by a pH of 11.0 for 12 h, were investigated. During the recovery phase, the performance, anaerobic ammonia oxidation (anammox) activity, microbial community, and correlation of bacteria as well as the influencing factors were evaluated synchronously. The performance of the CANON process deteriorated rapidly with a nitrogen removal rate (NRR) of 0.13 kg·m -3 ·d -1 , and Firmicutes, spore-forming bacteria, were the dominant phyla after alkaline shock. However, it could self-restore within 107 days after undergoing four stages, at which Planctomycetes became dominant with a relative abundance of 64.62%. Network analysis showed that anammox bacteria ( Candidatus Jettenia , Kuenenia , and Brocadia ) were positively related to some functional bacteria such as Nitrosomonas , SM1A02 , and Calorithrix . Canonical correspondence analysis presented a strong correlation between the microbial community and influencing factors during the recovery phase. With the increase of nitrogen loading rate, the decrease of free nitrous acid and the synergistic effects, heme c content, specific anammox activity (SAA), NRR, and the abundance of dominant genus increased correspondingly. The increase of heme c content regulates the quorum sensing system, promotes the secretion of extracellular polymeric substances, and further improves SAA, NRR, and the relative abundance of the dominant genus. This study highlights some implications for the recovery of the CANON reactor after being exposed to an alkaline shock., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Ruili Yang et al.)

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd 1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d 1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d 1 -bound form. The heme d 1 -free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d 1 -binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed., (open access.)

Background: Stroke is one of the largest problems and clinical-social challenges within neurology and, in general, pathology. Here, we briefly reviewed the main pathophysiological mechanisms of ischemic stroke, which represent targets for medical interventions, including for a calf blood deproteinized hemodialysate/ultrafiltrate., Methods: We conducted a systematic review of current related literature concerning the effects of Actovegin ® , of mainly the pleiotropic type, applied to the injury pathways of ischemic stroke., Results: The bibliographic resources regarding the use of Actovegin ® in ischemic stroke are scarce. The main Actovegin ® actions refer to the ischemic stroke lesion items' ensemble, targeting tissue oxidation, energy metabolism, and glucose availability through their augmentation, combating ischemic processes and oxidative stress, and decreasing inflammation (including with modulatory connotations, by the nuclear factor-κB pathway) and apoptosis-like processes, counteracting them by mitigating the caspase-3 activation induced by amyloid β-peptides., Conclusion: Since no available therapeutic agents are capable of curing the central nervous system's lesions, any contribution, such as that of Actovegin ® (with consideration of a positive balance between benefits and risks), is worthy of further study and periodic reappraisal, including investigation into further connected aspects.

Sirohaem is a modified tetrapyrrole and a key prosthetic group of several enzymes involved in nitrogen and sulfur metabolisms. This work shows that Staphylococcus aureus produces sirohaem through a pathway formed by three independent enzymes. Of the two putative sirohaem synthases encoded in the S. aureus genome and annotated as cysG, one is herein shown to be a uroporphyrinogen III methyltransferase that converts uroporphyrinogen III to precorrin-2, and was renamed as UroM. The second cysG gene encodes a precorrin-2 dehydrogenase that converts precorrin-2 to sirohydrochlorin, and was designated as P2D. The last step was found to be performed by the gene nirR that, in fact, codes for a protein with sirohydrochlorin ferrochelatase activity, labelled as ShfC. Additionally, site-directed mutagenesis studies of S. aureus ShfC revealed that residues H22 and H87, which are predicted by homology modelling to be located at the active site, control the ferrochelatase activity. Within bacteria, sirohaem synthesis may occur via one, two or three enzymes, and we propose to name the correspondent pathways as Types 1, 2 and 3, respectively. A phylogenetic analysis revealed that Type 1 is the most used pathway in Gammaproteobacteria and Streptomycetales, Type 2 predominates in Fibrobacteres and Vibrionales, and Type 3 predominates in Firmicutes of the Bacillales order. Altogether, we concluded that the current distribution of sirohaem pathways within bacteria, which changes at the genus or species level and within taxa, seems to be the result of evolutionary multiple fusion/fission events., (© 2019 Federation of European Biochemical Societies.)

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC., (open access.)

In nature, sulfur exists in a range of oxidation states and the two-electron reduced form is the most commonly found in biomolecules like the sulfur-containing amino acids cysteine and methionine, some cofactors, and polysaccharides. Sulfur is reduced through two pathways: dissimilation, where sulfite (SO2-3) is used as terminal electron acceptor; and assimilation, where sulfite is reduced to sulfide (S2-) for incorporation into biomass. The pathways are independent, but share the sulfite reductase function, in which a single enzyme reduces sulfite by six electrons to make sulfide. With few exceptions, sulfite reductases from either pathway are iron metalloenzymes with structurally diverse configurations that range from monomers to tetramers. The hallmark of sulfite reductase is its catalytic center made of an iron-containing porphyrinoid called siroheme that is covalently coupled to a [4Fe-4S] cluster through a shared cysteine ligand. The substrate evolves through a push-pull mechanism, where electron transfer is coupled to three dehydration steps. Siroheme is an isobacteriochlorin that is more readily oxidized than protoporphyin IX-derived hemes. It is synthesized from uroporphyrinogen III in three steps (methylation, a dehydrogenation, and ferrochelation) that are performed by enzymes with homology to those involved in cobalamin synthesis. Future research will need to address how the siroheme-[4Fe-4S] clusters are assembled into apo-sulfite and nitrite reductases. The chapter will discuss how environmental microbes use sulfite reductase to survive in a range of ecosystems; how atomic-resolution structures of dissimilatory and assimilatory sulfite reductases reveal their ancient homology; how the siroheme-[4Fe-4S] cluster active site catalyzes the six-electron reduction of sulfite to sulfide; and how siroheme is synthesized across diverse microrganisms.

Skeletal muscle injuries in competitive sports cause lengthy absences of athletes from tournaments. This is of tremendous competitive and economic relevance for both the athletes and their respective clubs. Therapy for structural muscle lesions aims to promote regeneration and fast-track return-to-play. A common clinical treatment strategy for muscle injuries is the intramuscular injection of calf blood compound and the homeopathic drug, Tr14. Although the combination of these two agents was reported to reduce recovery time, the regulatory mechanism whereby this occurs remains unknown. In this in vivo study, we selected a rat model of mechanical muscle injury to investigate the effect of this combination therapy on muscle regeneration. Gene expression analysis and histological images revealed that this combined intramuscular injection for muscle lesions can enhance the expression of pro-myogenic genes and proteins and accelerate muscle regeneration. These findings are novel and depict the positive effects of calf blood compound and the homeopathic drug, Tr14, which are utilized in the field of Sports medicine.

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD + -dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of siroheme synthesis.

HutZ from Vibrio cholerae (VcHutZ) is a dimeric protein that catalyzes oxygen-dependent degradation of heme. The reaction mechanism is the same as that of canonical heme oxygenase (HO), but the structure of HutZ is quite different from that of HO. Thus, we postulate that HutZ has evolved via a different pathway from that of HO. The Alr5027 protein from cyanobacteria possessing proteins potentially related to ancestral proteins utilizing O 2 in enzymatic reactions is homologous to HutZ family proteins (67% similarity), but the heme axial ligand of HutZ is not conserved in Alr5027. To investigate whether Alr5027 can bind and degrade heme, we expressed Alr5027 in Escherichia coli and purified it. Although Alr5027 did not bind heme, replacement of Lys164, corresponding to the heme axial ligand of HutZ, with histidine conferred heme-binding capability. The K164H mutant produced verdoheme in the reaction with H 2 O 2 , indicating acquisition of heme-degradation ability. Among the mutants, the K164H mutant produced verdoheme most efficiently. Although the K164H mutant did not degrade heme through ascorbic acid, biliverdin, the final product of VcHutZ, was formed by treatment of verdoheme with ascorbic acid. An analysis of Trp103 fluorescence indicated elongation of the distance between protomers in this mutant compared with VcHutZ-the probable cause of the inefficiency of ascorbic acid-supported heme-degradation activity. Collectively, our findings indicate that a single lysine-to-histidine mutation converted Alr5027 to a heme-binding protein that can form verdoheme through H 2 O 2 , suggesting that HutZ family proteins have acquired the heme-degradation function through molecular evolution from an ancestor protein of Alr5027., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The active site of sulfite reductase (SiR) consists of an unusual siroheme-Fe 4 S 4 assembly coupled via a cysteinate sulfur, and serves for multi-electron reduction reactions. Clear explanations have not been demonstrated for the reasons behind the choice of siroheme (vs. other types of heme) or for the single-atom coupling to an Fe 4 S 4 center (as opposed to simple adjacency or to coupling via chains consisting of more than one atom). Possible explanations for these choices have previously been invoked, relating to the control of the spin state of the substrate-binding (siro)heme iron, modulation of the trans effect of the (Fe 4 S 4 -bound) cysteinate, or modulation of the redox potential. Reported here is a density functional theory (DFT) investigation of the structural interplay (in terms of geometry, molecular orbitals and magnetic interactions) between the siroheme and the Fe 4 S 4 center as well as the importance of the covalent modifications within siroheme compared to the more common heme b, aiming to verify the role of the siroheme modification and of the Fe 4 S 4 cluster at the SiR active site, with focus on previously-formulated hypotheses (geometrical/sterics, spin state, redox and electron-transfer control). A calibration of various DFT methods/variants for the correct description of ground state spin multiplicity is performed using a set of problematic cases of bioinorganic Fe centers; out of 11 functionals tested, M06-L and B3LYP offer the best results - though none of them correctly predict the spin state for all test cases. Upon examination of the relative energies of spin states, reduction potentials, energy decomposition (electrostatic, exchange-repulsion, orbital relaxation, correlation and dispersion interactions) and Mayer bond indices in SiR models, the following main roles of the siroheme and cubane are identified: (1) the cubane cofactor decreases the reduction potential of the siroheme and stabilizes the siroheme-cysteine bond interaction, and (2) the siroheme removes the quasi-degeneracy between the intermediate and high-spin states found in ferrous systems by preserving the latter as ground state; the higher-spin preference and the increased accessibility of multiple spin states are likely to be important in selective binding of the substrate and of the subsequent reaction intermediates, and in efficient changes in redox states throughout the catalytic cycle., 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 © 2019 Elsevier Inc. All rights reserved.)

Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca 2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent-response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance., Competing Interests: The authors declare no competing interest.

Cytochrome c oxidase (CcO), the Cu A, heme a, heme a 3 , Cu B enzyme of respiratory chain, converts the free energy released by aerobic cytochrome c oxidation into a membrane electrochemical proton gradient (ΔμH + ). ΔμH + derives from the membrane anisotropic arrangement of dioxygen reduction to two water molecules and transmembrane proton pumping from a negative (N) space to a positive (P) space separated by the membrane. Spectroscopic, potentiometric, and X-ray crystallographic analyses characterize allosteric cooperativity of dioxygen binding and reduction with protonmotive conformational states of CcO. These studies show that allosteric cooperativity stabilizes the favorable conformational state for conversion of redox energy into a transmembrane ΔμH + ., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Actovegin is a biological drug with a controversial history of use in the treatment of sports injuries during the past 60 years. Particular concerns have been raised about its ergogenic potential to enhance performance, but some of these have been based on little more than anecdote., Objectives: In this article, we review the most recent scientific evidence to determine the clinical efficacy, safety profile, and legal status of Actovegin., Methods: We considered all studies directly commenting on experience with Actovegin use as the primary intervention within the past 10 years. Outcomes included mechanisms of action, clinical efficacy in enhancing muscle repair, any report of safety issues, and any evidence for ergogenic effect., Results: Our database search returned 212 articles, abstracts were screened, and after inclusion/exclusion criteria were applied, 25 articles were considered: Publications included 11 primary research articles (7 in vitro studies and 4 clinical trials), 8 review articles, 5 editorials, and a single case report., Conclusions: Current literature is still yet to define the active compound(s) of Actovegin, but suggests that it shows antioxidant and antiapoptotic properties, and may also upregulate macrophage responses central to muscle repair. Clinical efficacy was supported by one new original research article, and the use of Actovegin to treat muscle injuries remains safe and supported. Two articles argued the ergogenic effect of Actovegin, but in vitro findings did not to translate to the outcomes of a clinical trial. An adequate and meaningful scientific approach remains difficult in a field where there is immense pressure to deliver cutting-edge therapies.

Objective: To compare the effects of cortexin, cerebrolysin and actovegin on memory impairment, cerebral circulation and morphological changes in the hippocampus of rats with chronic brain ischemia., Material and Methods: The study was conducted using male rats with chronic brain ischemia caused by stenosis of the common carotid arteries by 50%. Animals received cortexin (0,3; 1 or 3 mg/kg), cerebrolysin (0,8; 2,5 or 7,5 ml/kg) and actovegin (5 ml/kg) in two 10-day courses with 10 days of treatment break. The severity of cognitive impairment was evaluated using the Morris water maze, passive and active avoidance tests. Cerebral circulation using laser flowmetry and brain hippocampus structures were studied in the end of treatment., Results: Cognitive impairment in animals with chronic brain ischemia was accompanied by the development of pathological changes in the CA1 and CA4 regions of the hippocampus. Administration of cortexin (1 and 3 mg/kg) and cerebrolysin (2.5 and 7.5 ml/kg) to rats with chronic brain ischemia had almost no effect on cerebral blood flow, but contributed to the improvement in memory formation and retrieval processes in the Morris water maze. The treatment effect was comparable for both drugs and persisted after 10 days of treatment break. Morphological assessment showed a decrease in the severity of pathological changes in the hippocampal regions., Conclusion: The course-administration of cortexin and cerebrolysin lead to a decrease in the severity of memory impairment and pathomorphological changes in the hippocampus in rats with chronic brain ischemia.

The use of injections to treat structural muscle injuries is controversially discussed. In our controlled in vitro study, we investigated the biological impact of Actovegin and Traumeel alone and in combination on primary human skeletal muscle cells. Cells were characterized by immunofluorescence staining for myogenic factor 5 (Myf5) and MyoD, and cultured with or without Actovegin and / or Traumeel. The effects of these agents were assayed by cell viability and gene expression of the specific markers MyoD, Myf5, neural adhesion molecule (NCAM), and CD31. Myotube formation was determined by myosin staining. Neither Actovegin nor Traumeel showed toxic effects or influenced cell viability significantly. High volumes of Actovegin down-regulated gene expression of NCAM after 3 days but had no effect on MyoD, Myf5, and CD31 gene expression. High volumes of Traumeel inhibited MyoD gene expression after 3 days, whereas after 7 days MyoD expression was significantly up-regulated. The combination of both agents did not significantly influence cell viability or gene expression. This is the first study demonstrating that Actovegin and Traumeel potentially modulate human skeletal muscle cells. The relevance of these in vitro findings has to be highlighted in further in vivo studies., Competing Interests: Authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Secondary coordination sphere interactions are critical in facilitating the formation, stabilization, and enhanced reactivity of high-valent oxidants required for essential biochemical processes. Herein, we compare the C-H bond oxidizing capabilities of spectroscopically characterized synthetic heme iron(IV) oxo complexes, F 8 Cmpd-II (F 8 = tetrakis(2,6-difluorophenyl)porphyrinate), and a 2,6-lutidinium triflate (LutH + ) Lewis acid adduct involving ferryl O-atom hydrogen-bonding, F 8 Cmpd-II(LutH + ). Second-order rate constants utilizing C-H and C-D substrates were obtained by UV-vis spectroscopic monitoring, while products were characterized and quantified by EPR spectroscopy and gas chromatography (GC). With xanthene, F 8 Cmpd-II(LutH + ) reacts 40 times faster ( k 2 = 14.2 M -1 s -1 ; -90 °C) than does F 8 Cmpd-II, giving bixanthene plus xanthone and the heme product [F 8 Fe III OH 2 ] + . For substrates with greater C-H bond dissociation energies (BDEs) F 8 Cmpd-II(LutH + ) reacts with the second order rate constants k 2 (9,10-dihydroanthracene; DHA) = 0.485 M -1 s -1 and k 2 (fluorene) = 0.102 M -1 s -1 (-90 °C); by contrast, F 8 Cmpd-II is unreactive toward these substrates. For xanthene vs xanthene-( d 2 ), large, nonclassical deuterium kinetic isotope effects are roughly estimated for both F 8 Cmpd-II and F 8 Cmpd-II(LutH + ). The deuterated H-bonded analog, F 8 Cmpd-II(LutD + ), was also prepared; for the reaction with DHA, an inverse KIE (compared to F 8 Cmpd-II(LutH + )) was observed. This work originates/inaugurates experimental investigation of the reactivity of authentic H-bonded heme-based Fe IV ═O compounds, critically establishing the importance of oxo H-bonding (or protonation) in heme complexes and enzyme active sites.

Many bacteria can switch from oxygen to nitrogen oxides, such as nitrate or nitrite, as terminal electron acceptors in their respiratory chain. This process is called "denitrification" and enables biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa, making it more resilient to antibiotics and highly adaptable to different habitats. The reduction of nitrite to nitric oxide is a crucial step during denitrification. It is catalyzed by the homodimeric cytochrome cd 1 nitrite reductase (NirS), which utilizes the unique isobacteriochlorin heme d 1 as its reaction center. Although the reaction mechanism of nitrite reduction is well understood, far less is known about the biosynthesis of heme d 1 . The last step of its biosynthesis introduces a double bond in a propionate group of the tetrapyrrole to form an acrylate group. This conversion is catalyzed by the dehydrogenase NirN via a unique reaction mechanism. To get a more detailed insight into this reaction, the crystal structures of NirN with and without bound substrate have been determined. Similar to the homodimeric NirS, the monomeric NirN consists of an eight-bladed heme d 1 -binding β-propeller and a cytochrome c domain, but their relative orientation differs with respect to NirS. His147 coordinates heme d 1 at the proximal side, whereas His323, which belongs to a flexible loop, binds at the distal position. Tyr461 and His417 are located next to the hydrogen atoms removed during dehydrogenation, suggesting an important role in catalysis. Activity assays with NirN variants revealed the essentiality of His147, His323 and Tyr461, but not of His417., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The spin state transition from low spin to high spin upon substrate addition is one of the key steps in cytochrome P450 catalysis. External perturbations such as pH and hydrogen bonding can also trigger the spin state transition of hemes through deprotonated histidine (e.g. Cytochrome c). In this work, we report the isolated 2-methylimidazole Cobalt(II) [Co(TPP)(2-MeHIm)] and [Co(TTP)(2-MeHIm)], and the corresponding 2-methylimidazolate derivatives where the N-H proton of axial 2-MeHIm is removed. Interestingly, various spectroscopies including EPR and XAFS determine a high-spin state (S = 3/2) for the imidazolate derivatives, in contrast to the low-spin state (S = 1/2) of all known imidazole analogs. DFT assisted stereoelectronic investigations are applied to understand the metal-ligand interactions, which suggest that the dramatically displaced metal center allowing a promotion e g (d π ) → b 1g ([Formula: see text]) is crucial for the occurrence of the spin state transition.

Here, we report the preparation and photo-physical characterization of hexa-coordinated vertebrate globins, human neuroglobin (hNgb) and cytoglobin (hCygb), with the native iron protoporphyrin IX (FePPIX) cofactor replaced by a fluorescent isostructural analogue, zinc protoporphyrin IX (ZnPPIX). To facilitate insertion of ZnPPIX into hexa-coordinated globins, apoproteins prepared via butanone extraction were unfolded by the addition of GuHCl and subsequently slowly refolded in the presence of ZnPPIX. The absorption/emission spectra of ZnPPIX reconstituted hCygb are similar to those observed for ZnPPIX reconstituted myoglobin whereas the absorption and emission spectra of ZnPPIX reconstituted hNgb are blue shifted by ∼2 nm. Different steady state absorption and emission properties of ZnPPIX incorporated in hCygb and hNgb are consistent with distinct hydrogen bonding interactions between ZnPPIX and the globin matrix. The fluorescence lifetime of ZnPPIX in hexa-coordinated globins is bimodal pointing towards increased heterogeneity of the heme binding cavity in hCygb and hNgb. ZnPPIX reconstituted Ngb binds to cytochrome c with the same affinity as reported for the native protein, suggesting that fluorescent analogues of Cygb and Ngb can be readily employed to monitor interactions between vertebrate hexa-coordinated globins and other proteins.

Psoriasis is a chronic recurrent skin inflammatory disease, and inhibition of inflammation may be an effective means of treating psoriasis. The flavonoid genistein has a clear anti-inflammatory effect. However, the anti-psoriatic effects of genistein and their underlying mechanisms remain unclear. In this study, we investigated the effects of genistein on imiquimod (IMQ)-induced psoriasis-like skin lesions in vivo and explored the mechanisms underlying those effects in vitro. It was found that genistein can significantly improve IMQ-induced pathological scores of cutaneous skin lesions in mice, reduce epidermal thickness, and inhibit the expression of inflammatory factors，including interleukin (IL)-1β, IL-6, tumour necrosis factor-alpha (TNF-α), chemokine ligand 2 (CCL2), IL-17 and IL-23. In vitro studies, genistein inhibited the proliferation of human keratinocyte HaCaT cells and inhibited the expression of inflammatory factors in a dose-dependent manner which induced by TNFα. Further researches showed that genistein could also significantly inhibit phosphorylated STAT3 (pSAT3) expression in IMQ mice dorsal skin and in TNF-α-induced HaCaT cells. The inhibitory effect of genistein on the expression of IL-6, IL-23 and TNF-α was weakened after Stat3 siRNA in HaCaT cells. Genistein could also significantly inhibit TNF-α induced the nuclear translocation of NF-κB, and inhibit the phosphorylation of I-kBα (pI-kBα). After combining with NF-κB blocker BAY 11-7082, the effect of genistein down-regulate the expression of TNF-α and VEGFA was attenuated in HaCaT cells. The results suggest that genistein may be developed for the treatment of psoriasis lesions., (Copyright © 2019. Published by Elsevier B.V.)

The characteristic carmine red color due to heme proteins is always observed in enriched anammox biomass. Heme c is a very important co-factor participating the main metabolic reactions with catalytic and electron-transfer potential in the anammox bacteria, and is possible for use as an indicator to evaluate anammox performance. Knowledge of the relationship between the heme c concentration and the anammox reactor performance is, however, very limited available information is constrained at an operation temperature of 35 °C. In this study, we report the heme c concentration change along with nitrogen removal rate (NRR) in three anammox expanded granular sludge bed reactors operated at different temperatures (15, 25, 35 °C). The response of specific anammox activity (SAA) to temperature was revealed for biomass originating from three reactors. The results indicate a strong relationship between heme c concentration and NRR at different culture temperatures. The possibility of evaluating the anammox performance by combining heme c quantification and the temperature is revealed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pathways formed of delocalized π-electron systems and polar groups of polypeptide chains bridged by hydrogen bonds are referred as π-H pathways. Suitable for electron transfer, these pathways in cytochrome c oxidase connect CuA, the source of electrons distributed in cytochrome c oxidase, with the metal centers, heme a, heme a 3 , CuB, the constituents of the catalytic binuclear center. The unusually rapid electron transfer between heme a and heme a 3 would have been facilitated by the link pathway of a long sequence of alternate peptide unit and hydrogen bond spanning Pro336-Val374, referred as suprahelix, between these hemes. Two pathways between CuA center and zinc center, share some portions with purported proton-translocating channels, designated "K" and "D"., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O 2 . Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b 3 and non-heme Fe B , respectively. The determined sensitivity (-4.00 × 10 -8  ± 1.84 × 10 -9  A/μM and - 2.71 × 10 -8  ± 1.44 × 10 -9  A/μM for NO and O 2 , respectively), limit of detection (0.5 μM for NO and 1.0 μM for O 2 ) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O 2 , respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O 2 , while the O 2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A variety of heme derivatives are pervasive in nature, having different architectures that are complementary to their function. Herein, we report the synthesis of a series of iron porphyrinoids, which bear electron-withdrawing groups and/or are saturated at the β-pyrrolic position, mimicking the structural variation of naturally occurring hemes. The effects of the aforementioned factors were systematically studied using a combination of electrochemistry, spectroscopy, and theoretical calculations with the carbon monoxide (CO) and nitric oxide (NO) adducts of these iron porphyinoids. The reduction potentials of iron porphyrinoids vary over several hundreds of millivolts, and the X-O (X = C, N) vibrations of the adducts vary over 10-15 cm -1 . Density functional theory calculations indicate that the presence of electron-withdrawing groups and saturation of the pyrrole ring lowers the π*-acceptor orbital energies of the macrocycle, which, in turn, attenuates the bonding of iron to CO and NO. A hypothesis has been presented as to why cytochrome c containing nitrite reductases and cytochrome cd 1 containing nitrite reductases follow different mechanistic pathways of nitrite reduction. This study also helps to rationalize the choice of heme a 3 and not the most abundant heme b cofactor in cytochrome c oxidase.

Aim: To assess the plasma level of N-acetylaspartate (NAA) before and after combined therapy with antidepressants and actovegin in a group of elderly patients diagnosed with depression., Material and Methods: Nineteen patients, 7 men and 12 women, mean age 70.5±5.8 years, were studied using clinical examination and psychometric scales as well as computed tomography (CT). NAA plasma levels were determined. The duration of treatment with antidepressants (venlafaxine, fluvoxamine) and actovegin was 28 days, patients were examined at baseline and on the 28 th day of treatment., Results and Conclusion: The NAA plasma level was reduced in patients compared to healthy volunteers. The increase of this indicator after treatment reflected a significant improvement on clinical and psychometric measures. The dynamics of NAA changes (increase or decrease) showed heterogeneity in the group of patients, which was not related to the efficacy of treatment but was correlated with comorbid diseases, in particular vascular diseases, and CT changes (leukoaraiosis). The authors consider the results of this study as preliminary.

Aim: The study of Actovegin effectin clinical presentations and hemorheological characteristics in patients with chronic cerebrovascular pathology (CCVP) and mild cognitive impairment., Materials and Methods: The study group included 47 patients (25 male and 22 female), aged 61-75 years (mean age 63.8±5.4) with CCVP who were treated with Actovegin. The control group comprised 28 patients matched by gender and age, without associated cerebrovascular pathology. All patients along with thorough neurological examination underwent laboratory analyses (platelet and erythrocyte rheology), neurovisualization studies (functional magnetic resonance imaging of the brain). Depending on the dosage all patients were divided in two groups: Actovegin 1000 mg and 160 mg daily., Results: Overall, with Actovegin treatment in 81% of cases positive dynamics both in subjective symptoms, and somatic status was observed. A favorable effect on cognitive function in patients with CCVP was noted. The dose-dependent drug effect was demonstrated. The effect of Actovegin on blood cell functioning included the formation of smaller (Tf and Ts; p=0.0096 and p=0.016) and less solid (γ dis) erythrocyte aggregates (p=0.0034) both in the study and control group. The increase in erythrocyte deformability during therapy was significantly associated with cognitive improvement (via MoCA test, r=0.28)., Conclusion: Complex (including neuropsychological and neurovisualizational) examination may not only help determine the cognitive status in patients with CCVP, but also assess the efficacy of neurometabolic therapy. New facts of Actovegin's influence on erythrocyte aggregation and deformability have been identified, which may enhance micro- and macrocirculation. The acquired data may prove the wide spectrum of Actovegin's pharmacological effect, which allows to use it in all forms of cerebrovascular pathology.