Actovegin improves skeletal muscle mitochondrial respiration and functional aerobic capacity in a type 1 diabetic male murine model.

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.

Actovegin in the management of patients after ischemic stroke: A systematic review.

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.

Heme A Synthase Deficiency Affects the Ability of Bacillus cereus to Adapt to a Nutrient-Limited Environment.

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.

Protonation of the oxo-bridged heme/copper assemblies: Modeling the oxidized state of the cytochrome c oxidase active site.

In this study on model compounds for the resting oxidized state of the iron­copper binuclear center in cytochrome c oxidase (CcO), we describe the synthesis of a new µ-oxo-heme/Cu complex, [(TPP)FeIII-O-CuII(tmpa)][B(C6F5)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 1H NMR and 19F 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 pKa values for the corresponding µ-hydroxo-heme/Cu species (i.e., the first protonation event) have been reported and compared with the pKa ranges previously estimated for related systems. These synthetic systems may represent structural models for the oxidized FeIII-X-CuII 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 Fea3-OHx-CuB moiety. Here, we investigated two successive protonation events, for a series of µ-oxo-heme/Cu assemblies and reported the pKa values for the first protonation event. The µ-aquo-heme/Cu complexes described here are the first examples of such systems.

Biosynthesis and trafficking of heme o and heme a: new structural insights and their implications for reaction mechanisms and prenylated heme transfer.

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.

[Antioxidant effect of cortexin, cerebrolysin and actovegin in rats with chronic cerebrovascular insufficiency]. / Antioksidantnoe deistvie Korteksina, Tserebrolizina i Aktovegina u zhivotnykh s khronicheskoi ishemiei golovnogo mozga.

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.

Beating Heart of Cytochrome c Oxidase: The Shared Proton of Heme a3 Propionates.

Cytochrome c oxidase (CcO) 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 CcO 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 CcO were suggested as PLS candidates although, according to CcO 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 a3 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 pKA values of all titratable residues in CcO. For the modeled structures, the heme a3 propionates have pKA values high enough to host a proton transiently but not too high to fix the proton permanently. The change in pKA 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.

Neuroprotective action of Cortexin, Cerebrolysin and Actovegin in acute or chronic brain ischemia in rats.

This study was the first to compare the neuroprotective activity of Cerebrolysin®, Actovegin® and Cortexin® in rodent models of acute and chronic brain ischemia. The neuroprotective action was evaluated in animals with acute (middle cerebral artery occlusion) or chronic (common carotid artery stenosis) brain ischemia models in male rats. Cortexin® (1 or 3 mg/kg/day), Cerebrolysin® (538 or 1614 mg/kg/day) and Actovegin® (200 mg/kg/day) were administered for 10 days. To assess the neurological and motor impairments, open field test, adhesive removal test, rotarod performance test and Morris water maze test were performed. Brain damage was assessed macro- and microscopically, and antioxidant system activity was measured in brain homogenates. In separate experiments in vitro binding of Cortexin® to a wide panel of receptors was assessed, and blood-brain barrier permeability of Cortexin® was assessed in mice in vivo. Cortexin® or Cerebrolysin® and, to a lesser extent, Actovegin® improved the recovery of neurological functions, reduced the severity of sensorimotor and cognitive impairments in rats. Cortexin® reduced the size of necrosis of brain tissue in acute ischemia, improved functioning of the antioxidant system and prevented the development of severe neurodegenerative changes in chronic ischemia model. Radioactively labeled Cortexin® crossed the blood-brain barrier in mice in vivo with concentrations equal to 6-8% of concentrations found in whole blood. During in vitro binding assay Cortexin® (10 µg/ml) demonstrated high or moderate binding to AMPA-receptors (80.1%), kainate receptors (73.5%), mGluR1 (49.0%), GABAA1 (44.0%) and mGluR5 (39.7%), which means that effects observed in vivo could be related on the glutamatergic and GABAergic actions of Cortexin®. Thus, Cortexin, 1 or 3 mg/kg, or Cerebrolysin®, 538 or 1614 mg/kg, were effective in models acute and chronic brain ischemia in rats. Cortexin® contains compounds acting on AMPA, kainate, mGluR1, GABAA1 and mGluR5 receptors in vitro, and readily crosses the blood-brain barrier in mice.

Repurposed and artificial heme enzymes for cyclopropanation reactions.

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.

Implication of a Key Region of Six Bacillus cereus Genes Involved in Siroheme Synthesis, Nitrite Reductase Production and Iron Cluster Repair in the Bacterial Response to Nitric Oxide Stress.

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.

Pathways of Iron and Sulfur Acquisition, Cofactor Assembly, Destination, and Storage in Diverse Archaeal Methanogens and Alkanotrophs.

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 F430) 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.

Ionic bond in hydrogen transferring of the ferrous and/or ferric human/mouse verdoheme oxygenase.

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 HOMOFRVMO-LUMOImidazole 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 (Eads) 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.

Probing the role of Val228 on the catalytic activity of Scytalidium catalase.

Scytalidium catalase is a homotetramer including heme d in each subunit. Its primary function is the dismutation of H2O2 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.

A noncanonical heme oxygenase specific for the degradation of c-type heme.

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 ß- 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/ß 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.

[Therapeutic effect of Actovegin on acute oral mucositis caused by radiotherapy].

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-ß1 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-ß1, 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-ß1, and TNF-α in both groups was significantly lower than that before treatment (P<0.05). The serum level of CRP, IL-6, TGF-ß1 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.

Crystal structure of a photosynthetic LH1-RC in complex with its electron donor HiPIP.

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.

YtkA (CtaK) and YozB (CtaM) function in the biogenesis of cytochrome c oxidase in Bacillus subtilis.

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 aa3 and the cytochrome c oxidase cytochrome caa3 . 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 CuA center in cytochrome caa3 and seems to be a functional analogue to proteins of the periplasmic CuA chaperone family (PCuA C). CtaM is required for the activity of both cytochrome caa3 and cytochrome aa3 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.

Crystal structure of NirF: insights into its role in heme d1 biosynthesis.

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 d1 . While heme d1 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 d1 , the last intermediate of heme d1 biosynthesis. We found that NirF forms a bottom-to-bottom ß-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 d1 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 d1 derivatives. DATABASE: Structural data are available in PDB database under the accession numbers 6TV2 and 6TV9.

Heme P460: A (Cross) Link to Nitric Oxide.

Ammonia-oxidizing bacteria (AOB) convert ammonia (NH3) to nitrite (NO2-) as their primary metabolism and thus provide a blueprint for the use of NH3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH2OH) to NO2-. 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 NH2OH and were both originally reported to produce NO2-. 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 NH2OH oxidation. Under aerobic conditions substoichiometric production of NO2- was observed along with production of nitrous oxide (N2O). Under anaerobic conditions, however, N2O was the exclusive product of NH2OH 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 O2, thus producing NO2- abiotically. Because N2O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO2- is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO2- 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 NH2OH 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 NH2OH. 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.

[Comparative study of protective effects of Cortexin, Cerebrolysin and Actovegin on memory impairment, cerebral circulation and morphological changes in the hippocampus of rats with chronic brain ischemia]. / Sravnitel'noe izuchenie vliyaniya Korteksina, Tserebrolizina i Aktovegina na sostoyanie pamyati, mozgovoe krovoobrashchenie i strukturu gippokampa u krys s khronicheskim narusheniem mozgovogo krovoobrashcheniya.

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.