Search

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation., Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council, Depto. de Química Física, Fac. de Ciencias Químicas, TRUE, pub

In blood circulation, the complement and the coagulation cascades, together with platelets and endothelial cells form a complex network of crosstalk. When dysregulated, these interactions can lead to inflammation in combination with thrombosis (thromboinflammation) and the manifestation of pathophysiological complications. As complement activation and thromboinflammation are often associated with intravascular hemolysis, e.g., sickle cell disease (SCD), we aimed to study these reactions in relation to heme, a product of hemolysis. Furthermore, our goal was to evaluate whether exposure to biomaterials results in hemolysis-induced thromboinflammation, and to examine the potential of complement inhibition. Our findings show that heme could lead to a significant thromboinflammatory response in our in vitro whole blood model, as seen by complement-, cell- and coagulation- activation, as well as increased cytokine secretion. Inflammation, including complement activation, was also linked with increased heme concentrations in vivo in hemolytic disease in SCD patients. The mechanism of action was attributed to uncontrolled alternative pathway (AP) activation, as heme was shown to bind and inhibit the main AP regulator, factor I, resulting in increased concentrations of fluid phase and surface-bound C3b. Moreover, administration of iron oxide nanoparticles (IONPs) in vitro and implantation of left ventricular assist device (LVAD) in vivo were monitored and correlated with increased hemolytic, e.g., heme, and thromboinflammatory markers, e.g., complement-, endothelial cell- and platelet- activation. Targeting complement components C5 and C3 in vitro was shown overall beneficial in the presence of heme or IONPs respectively. In our settings, the majority of the thromboinflammatory markers measured were successfully attenuated, indicating that complement fuels this response. In conclusion, the results in this thesis stress that heme-induced complement activation is an important

Heme-copper oxidases (HCOs) utilize tyrosine (Tyr) to donate one of the four electrons required for the reduction of O2 to water in biological respiration, while tryptophan (Trp) is speculated to fulfill the same role in cyt bd oxidases. We previously engineered myoglobin into a biosynthetic model of HCOs and demonstrated the critical role that Tyr serves in the oxygen reduction reaction (ORR). To address the roles of Tyr and Trp in these oxidases, we herein report the preparation of the same biosynthetic model with the Tyr replaced by Trp and further demonstrate that Trp can also promote the ORR, albeit with lower activity. An X-ray crystal structure of the Trp variant shows a hydrogen-bonding network involving two water molecules that are organized by Trp, similar to that in the Tyr variant, which is absent in the crystal structure with the native Phe residue. Additional electron paramagnetic resonance measurements are consistent with the formation of a Trp radical species upon reacting with H2O2. We attribute the lower activity of the Trp variant to Trp's higher reduction potential relative to Tyr. Together, these findings demonstrate, for the first time, that Trp can indeed promote the ORR and provides a structural basis for the observation of varying activities. The results support a redox role for the conserved Trp in bd oxidase while suggesting that HCOs use Tyr instead of Trp to achieve higher reactivity.

Intravascular hemolysis results in the release of cell-free hemoglobin and heme in plasma. In sickle cell disease, the fragility of the sickle red blood cell leads to chronic hemolysis, which can contribute to oxidative damage and activation of inflammatory pathways. The scavenger proteins haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from the circulation. Heme also intercalates in membranes of blood cells and endothelial cells in the vasculature and associates with other plasma components such as albumin and lipoproteins. Hemopexin has a much higher affinity and can strip heme from the other pools and detoxify plasma from cell-free circulatory heme. However, due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. Thus, cell-free unbound heme is expected to accumulate in plasma. We developed a methodology for the accurate quantification of the fraction of heme, which is pathologically relevant in sickle cell disease, that does not appear to be sequestered to a plasma compartment. Our data show significant variation in the concentration of unbound heme, and rather unexpectedly, the size of the unbound fraction does not correlate to the degree of hemolysis, as measured by the concentration of bound heme. Very high heme concentrations (>150 µM) were obtained in some plasma with unbound concentrations that were several fold lower than in plasma with much lower hemolysis (<50 µM). These findings underscore the long-term effects of chronic hemolysis on the blood components and of the disruption of the essential equilibrium between release of hemoproteins/heme in the circulation and adaptative response of the scavenging/removal mechanisms. Understanding the clinical implications of this loss of response may provide insights into diagnostic and therapeutic targets in patients with sickle cell disease.

During photoperiodic growth, the light-dependent nature of chlorophyll synthesis in angiosperms necessitates robust control of the production of 5-aminolevulinic acid (ALA), the rate-limiting step in the initial stage of tetrapyrrole biosynthesis (TBS). We are interested in dissecting the post-translational control of this process, which suppresses ALA synthesis for chlorophyll synthesis in dark-grown plants. Using biochemical approaches for analysis of Arabidopsis wild-type (WT) and mutant lines as well as complementation lines, we show that the heme-synthesizing ferrochelatase 2 (FC2) interacts with protochlorophyllide oxidoreductase and the regulator FLU which both promote the feedback-controlled suppression of ALA synthesis by inactivation of glutamyl-tRNA reductase, thus preventing excessive accumulation of potentially deleterious tetrapyrrole intermediates. Thereby, FC2 stabilizes POR by physical interaction. When the interaction between FC2 and POR is perturbed, suppression of ALA synthesis is attenuated and photoreactive protochlorophyllide accumulates. FC2 is anchored in the thylakoid membrane via its membrane-spanning CAB (chlorophyll-a-binding) domain. FC2 is one of the two isoforms of ferrochelatase catalyzing the last step of heme synthesis. Although FC2 belongs to the heme-synthesizing branch of TBS, its interaction with POR potentiates the effects of the GluTR-inactivation complex on the chlorophyll-synthesizing branch and ensures reciprocal control of chlorophyll and heme synthesis., Chinese Scholarship Council, Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659, Peer Reviewed

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition path

Abdominal aortic aneurysm (AAA) is a mostly silent vascular disease, with a rupture-related mortality rate exceeding 80%. To aid in future diagnosis and prognosis efforts, the studies included in the present thesis have explored the role of specific oxidative stress-related factors in the context of AAA using two population-based cohorts: AAA screening cohort of 65-year-old males (n = 421) and the Malmö diet and cancer study (MDCS) (n = 25252). In Paper I, global DNA methylation (enzyme-linked immunosorbent assay (ELISA)) and homocysteine (diagnostic enzymatic assay) levels were investigated in the AAA screening cohort. Increased global DNA methylation and homocysteine levels were observed in AAA, with positive linear associations with baseline aortic diameter. However, no significant association was found with AAA growth, suggesting different mechanisms in initiation and progression. Paper II characterizes a novel mitochondrial genetic landscape in AAA from 48 cases and 48 matched controls from the AAA screening cohort. Whole mitochondrial genome sequencing revealed differential mutational landscapes in AAA as compared to the non-AAA group. MtDNA mutational load was significantly elevated in AAA, particularly in the regulatory and conserved region (MT-TAS2). Lower mitochondrial copy number exacerbated the effect of mtDNA mutation in AAA. A novel 24 bp mtDNA duplication was found exclusive to AAA cases, and the northern-European JTU mitochondrial haplogroup was associated with family history of AAA. Paper III explores genetic variations in oxidative stress-related genes and antioxidant vitamin intake in incident intact AAA and ruptured AAA (rAAA) in the MDCS cohort. A variant in NOX3 gene was associated with higher rAAA risk, while antioxidant vitamins, riboflavin, and folate reduced intact AAA incidence. Both the studied genetic background and sex significantly modified the effect of specific vitamins’ intake on intact AAA risk ,overall, suggesting a complex gene-s

The unpleasant color caused by heme limits the utilization of hemoglobin as a food ingredient. Enzymatic hydrolysis has been used to decolorize hemoglobin, but the underlying mechanisms are poorly understood. The aim of this study was to investigate the decolorization efficiency of porcine hemoglobin using different enzymes and final pH values, and to elucidate their influence on decolorization. Based on higher yields and better decolorization, hemoglobin hydrolysates produced by papain, bromelain, savinase, and protease A were further studied. Compared to hydrolysates by savinase and protease A, a higher proportion of histidine-containing peptides was responsible for better decolorization by papain and bromelain. For all hydrolysates, a moderate reduction in pH to 4.0–5.0 facilitated decolorization of the hydrolysates. Similar peptide profiles of hydrolysates from the same enzyme treatment reflected that pH mainly affected the precipitation of the heme-containing fraction through heme–heme interaction rather than heme–peptide interaction. Overall, this study sheds light on the use of enzymatic hydrolysis to remove the heme group from hemoglobin. Practical Application: Slaughterhouses produce tons of protein-rich blood each year. Due to the presence of the heme group in hemoglobin, blood has a dark red color and metallic taste, making it generally unacceptable for consumers. This study provided information on the decolorization of porcine hemoglobin by removing the heme fraction, which should facilitate the utilization of decolored hemoglobin hydrolysates as nutritional food ingredients.

To meet the logistical needs of supply and demand for heme, organisms need to make it, move it to the right place, and degrade it when necessary. More specifically, the provision and distribution of heme must be coupled with the synthesis of heme proteins and meticulously regulated so that heme-tuned cellular pathways are only activated on demand. However, how cells handle heme to meet transient cellular requirements is yet to be clarified and represents a long-lasting question in bioinorganic chemistry. This study deploys a genetically encoded heme sensor (mAPXmEGFP) and Fluorescence Lifetime Imaging Microscopy to investigate the cellular mechanisms controlling heme bioavailability. The analysis is paired with the contextualisation of the data within the intricate network of regulation that oversees the levels of key proteins (i.e. ALAS1, HO-1, HO-2, and GAPDH) and their coordination with an intracellular heme buffering system that ensures heme homeostasis. Further, the implications and possible drawbacks deriving from the deployment of genetically encoded sensors in live cells is discussed and explored.

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

Cold shock protein D (CspD) is one of the homologous proteins of cold shock protein A (CspA), inhibiting DNA replication by binding to single-stranded DNA. We found that CspD from Vibrio cholerae (VcCspD) possesses one heme regulatory motif (HRM) sequence and specifically binds heme with a stoichiometry of 1:1. The binding of a synthetic single-stranded DNA oligomer (ssDNA) was followed by fluorescence quenching of Trp. The fluorescence quenching associated with the addition of ssDNA was suppressed in the presence of heme, indicating that heme binding to VcCspD inhibited the formation of the VcCspD-ssDNA complex. Such heme-induced inhibition was not observed for the VcCspD mutant with replacement of Cys22 in the HRM with alanine (C22A). Heme binding at Cys22 is, therefore, essential for the inhibition of ssDNA binding for VcCspD. The growth of Escherichia coli at 37 °C was slowed when VcCspD was overexpressed, indicating that VcCspD hampers the growth of E. coli. When the production of heme in cells was promoted by the addition of a heme precursor, d-aminolevulinic acid, the growth of E. coli expressing VcCspD was decelerated, but the growth of E. coli expressing the C22A mutant was not decelerated. These observations allow us to conclude that heme specifically binds to the HRM region in VcCspD and inhibits the binding of target ssDNA, which suggests that heme functions as a regulatory molecule for DNA replication.

Iron deficiency anemia can be treated with oral or intravenous Fe supplementation. Such supplementation has considerable effects on the human microbiome, and on opportunistic pathogenic micro-organisms. Molecular understanding of the control and regulation of Fe availability at the host-microbe interface is crucial to interpreting the side effects of Fe supplementation. Here, we provide a concise overview of the regulation of Fe by the opportunistic pathogen Staphylococcus aureus. Ferric uptake regulator (Fur) plays a central role in controlling Fe uptake, utilization and storage in order to maintain a required value. The micro-organism has a strong preference for heme iron as an Fe source, which is enabled by the Iron-regulated surface determinant (Isd) system. The strategies it employs to overcome Fe restriction imposed by the host include: hijacking host proteins, replacing metal cofactors, and replacing functions by non-metal dependent enzymes. We propose that integrated omics approaches, which include metalloproteomics, are necessary to provide a comprehensive understanding of the metal tug of war at the host-microbe interface down to the molecular level., RST/Applied Radiation & Isotopes, BT/Biocatalysis

Hemolysis, as a result of disease or exposure to biomaterials, is characterized by excess amounts of cell-free heme intravascularly and consumption of the protective heme-scavenger proteins in plasma. The liberation of heme has been linked to the activation of inflammatory systems, including the complement system, through alternative pathway activation. Here, we investigated the impact of heme on the regulatory function of the complement system. Heme dose-dependently inhibited factor I-mediated degradation of soluble and surface-bound C3b, when incubated in plasma or buffer with complement regulatory proteins. Inhibition occurred with factor H and soluble complement receptor 1 as co-factors, and the mechanism was linked to the direct heme-interaction with factor I. The heme-scavenger protein hemopexin was the main contaminant in purified factor I preparations. This led us to identify that hemopexin formed a complex with factor I in normal human plasma. These complexes were significantly reduced during acute vasoocclusive pain crisis in patients with sickle cell disease, but the complexes were normalized at their baseline outpatient clinic visit. Hemopexin exposed a protective function of factor I activity in vitro, but only when it was present before the addition of heme. In conclusion, we present a mechanistic explanation of how heme promotes uncontrolled complement alternative pathway amplification by interfering with the regulatory capacity of factor I. Reduced levels of hemopexin and hemopexin-factor I complexes during an acute hemolytic crisis is a risk factor for heme-mediated factor I inhibition.

Obstructive sleep apnea (OSA) is a common disorder associated with increased risk of cardiovascular disease and mortality. Iron and heme metabolism, implicated in ventilatory control and OSA comorbidities, was associated with OSA phenotypes in recent admixture mapping and gene enrichment analyses. However, its causal contribution was unclear. In this study, we performed pathway-level transcriptional Mendelian randomization (MR) analysis to investigate the causal relationships between iron and heme related pathways and OSA. In primary analysis, we examined the expression level of four iron/heme Reactome pathways as exposures and four OSA traits as outcomes using cross-tissue cis-eQTLs from the Genotype-Tissue Expression portal and published genome-wide summary statistics of OSA. We identify a significant putative causal association between up-regulated heme biosynthesis pathway with higher sleep time percentage of hypoxemia (p = 6.14 × 10-3). This association is supported by consistency of point estimates in one-sample MR in the Multi-Ethnic Study of Atherosclerosis using high coverage DNA and RNA sequencing data generated by the Trans-Omics for Precision Medicine project. Secondary analysis for 37 additional iron/heme Gene Ontology pathways did not reveal any significant causal associations. This study suggests a causal association between increased heme biosynthesis and OSA severity.

Three well-characterized heme peroxidases (cytochrome c peroxidase = CCP, ascorbate peroxidase = APX, and Leishmania major peroxidase = LMP) all have a Trp residue tucked under the heme stacked against the proximal His heme ligand. The reaction of peroxidases with H2O2 to give Compound I results in the oxidation of this Trp to a cationic radical in CCP and LMP but not in APX. Considerable experimental data indicate that the local electrostatic environment controls whether this Trp or the porphyrin is oxidized in Compound I. Attempts have been made to place the differences between these peroxidases on a quantitative basis using computational methods. These efforts have been somewhat limited by the approximations required owing to the computational cost of using fully solvated atomistic models with well-developed forcefields. This now has changed with available GPU computing power and the associated development of software. Here we employ thermodynamic integration and multistate Bennett acceptance ratio methods to help fine-tune our understanding on the energetic differences in Trp radical stabilization in all three peroxidases. These results indicate that the local solvent structure near the redox active Trp plays a significant role in stabilization of the cationic Trp radical.

Pathogenic Staphylococcus aureus actively acquires iron from human hemoglobin (Hb) using the IsdH surface receptor. Heme extraction is mediated by a tri-domain unit within the receptor that contains its second (N2) and third (N3) NEAT domains joined by a helical linker domain. Extraction occurs within a dynamic complex, in which receptors engage each globin chain; the N2 domain tightly binds to Hb, while substantial inter-domain motions within the receptor enable its N3 domain to transiently distort the globin's heme pocket. Using molecular simulations coupled with Markov modeling, along with stopped-flow experiments to quantitatively measure heme transfer kinetics, we show that directed inter-domain motions within the receptor play a critical role in the extraction process. The directionality of N3 domain motion and the rate of heme extraction is controlled by amino acids within a short, flexible inter-domain tether that connects the N2 and linker domains. In the wild-type receptor directed motions originating from the tether enable the N3 domain to populate configurations capable of distorting Hb's pocket, whereas mutant receptors containing altered tethers are less able to adopt these conformers and capture heme slowly via indirect processes in which Hb first releases heme into the solvent. Thus, our results show inter-domain motions within the IsdH receptor play a critical role in its ability to extract heme from Hb and highlight the importance of directed motions by the short, unstructured, amino acid sequence connecting the domains in controlling the directionality and magnitude of these functionally important motions.

This work presents a new method for determining iron-porphyrin-like complexes (Fe-Py) based on Continuous Flow Analysis (CFA) with chemiluminescence detection and its application to natural waters in an estuarine environment. The involved reaction is founded on luminol oxidation by hydrogen peroxide in the presence of Fe-Py complexes at pH 13. The detection limit is 7.2 pM hemin equivalent, the linear range extends to 150 nM and precision of the method is 6.4% at 0.25 nM (n = 8). This new method's detection limit is 15 times lower than the previous analytical procedure of Vong et al. (2007), based on Flow Injection Analysis (FIA) and using different chemical conditions. Moreover, the presented method is fast (90s/analysis), involves a low consumption of reagents, a small sample volume, and simplified sample handling. The method was applied to natural samples collected along the temperate macrotidal Aulne estuary (Bay of Brest, France). Here, we report for the first time on the spatial distribution of the Fe-Py complex concentration (dissolved, reactive particulate) over the entire salinity gradient of a macrotidal temperate estuary. The Fe-Py concentrations in the riverine and marine end-members were 0.873 ± 0.007 nM (S = 0.92) and 0.010 ± 0.004 nM (S = 34.86), respectively. Between these two salinities, non-conservative behaviour was observed, with an increase in Fe-Py concentrations to 1.142 ± 0.031 nM at S = 5.2 corresponding to the Maximum Turbidity Zone (MTZ), followed by a strong removal of Fe-Py in the salinity range 5–20. Then, the Fe-Py concentrations decreased linearly during mixing processes, reaching picomolar levels towards the coastal waters. The estimated entering flux from the river equaled 240 ± 2 g.d−1 whereas the net flux to coastal sea waters was 95 ± 10 g.d−1 leading to a loss of ~60%. The estuarine system globally acts as a sink for Fe-Py complexes, probably due to the aggregation of Fe-Py complexes on particles, to flocculation and/or sediment

The pathways for synthesizing tetrapyrroles, including heme and chlorophyll, are well-conserved among organisms, despite the divergence of several enzymes in these pathways. Protoporphyrinogen IX oxidase (PPOX), which catalyzes the last common step of the heme and chlorophyll biosynthesis pathways, is encoded by three phylogenetically-unrelated genes, hemY, hemG and hemJ. All three types of homologues are present in the cyanobacterial phylum, showing a mosaic phylogenetic distribution. Moreover, a few cyanobacteria appear to contain two types of PPOX homologues. Among the three types of cyanobacterial PPOX homologues, only a hemJ homologue has been experimentally verified for its functionality. An objective of this study is to provide experimental evidence for the functionality of the cyanobacterial PPOX homologues by using two heterologous complementation systems. First, we introduced hemY and hemJ homologues from Gloeobacter violaceus PCC7421, hemY homologue from Trichodesmium erythraeum, and hemG homologue from Prochlorococcus marinus MIT9515 into a Delta hemG strain of E. coli. hemY homologues from G. violaceus and T. erythraeum, and the hemG homologue of P. marinus complimented the E. coli strain. Subsequently, we attempted to replace the endogenous hemJ gene of the cyanobacterium Synechocystis sp. PCC6803 with the four PPOX homologues mentioned above. Except for hemG from P. marinus, the other PPOX homologues substituted the function of hemJ in Synechocystis. These results show that all four homologues encode functional PPOX. The transformation of Synechocystis with G. violaceus hemY homologue rendered the cells sensitive to an inhibitor of the HemY-type PPOX, acifluorfen, indicating that the hemY homologue is sensitive to this inhibitor, while the wild-type G. violaceus was tolerant to it, most likely due to the presence of HemJ protein. These results provide an additional level of evidence that G. violaceus contains two types of functional PPOX.

Hemorrhagic transformation of ischemic stroke has devastating consequences, with high mortality and poor functional outcomes. Animal models of ischemic stroke also demonstrate the potential for hemorrhagic transformation, which complicates biochemical characterization, treatment studies, and hinders poststroke functional outcomes in affected subjects. The incidence of hemorrhagic transformation of ischemic stroke in animal model research is not commonly reported. The postmortem brain of such cases presents a complex milieu of biomarkers due to the presence of healthy cells, regions of varying degrees of ischemia, dead and dying cells, dysregulated metabolites, and blood components (especially reactive Fe species released from lysed erythrocytes). To improve the characterization of hemorrhage biomarkers on an ischemic stroke background, we have employed a combination of histology, X-ray fluorescence imaging (XFI), and Fourier transform infrared (FTIR) spectroscopic imaging to assess 122 photothrombotic (ischemic) stroke brains. Rapid freezing preserves brain biomarkers in situ and minimizes metabolic artifacts due to postmortem ischemia. Analysis revealed that 25% of the photothrombotic models had clear signs of hemorrhagic transformation. The XFI and FTIR metabolites provided a quantitative method to differentiate key metabolic regions in these models. Across all hemorrhage cases, it was possible to consistently differentiate otherwise healthy tissue from other metabolically distinct regions, including the ischemic infarct, the ischemic penumbra, blood vessels, sites of hemorrhage, and a region surrounding the hemorrhage core that contained elevated lipid oxidation. Chemical speciation of deposited Fe demonstrates the presence of heme-Fe and accumulation of ferritin.

The protozoan parasite Leishmania, responsible for leishmaniasis, is one of the few aerobic organisms that cannot synthesize the essential molecule heme. Therefore, it has developed specialized pathways to scavenge it from its host. In recent years, some proteins involved in the import of heme, such as LHR1 and LFLVCRB, have been identified, but relevant aspects regarding the process remain unknown. Here, we characterized the kinetics of the uptake of the heme analogue Zn(II) Mesoporphyrin IX (ZnMP) in Leishmania major promastigotes as a model of a parasite causing cutaneous leishmaniasis with special focus on the force that drives the process. We found that ZnMP uptake is an active, inducible, and pH-dependent process that does not require a plasma membrane proton gradient but requires the presence of the monovalent cations Na+ and/or K+. In addition, we demonstrated that this parasite can efflux this porphyrin against a concentration gradient. We also found that ZnMP uptake differs among different dermotropic or viscerotropic Leishmania species and does not correlate with LHR1 or LFLVCRB expression levels. Finally, we showed that these transporters have only partially overlapping functions. Altogether, these findings contribute to a deeper understanding of an important process in the biology of this parasite.

Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for biosynthesis and drug metabolism. Monitoring the activity of CYP3A4, the major human drug-metabolizing enzyme, is vital for assessing the metabolism of pharmaceuticals and identifying harmful drug-drug interactions. Existing probes for CYP3A4 are irreversible turn-on substrates that monitor activity at specific time points in end-point assays. To provide a more dynamic approach, we designed, synthesized, and characterized emissive Ir(III) and Ru(II) complexes that allow monitoring of the CYP3A4 active-site occupancy in real time. In the bound state, probe emission is quenched by the active-site heme. Upon displacement from the active site by CYP3A4-specific inhibitors or substrates, these probes show high emission turn-on. Direct probe binding to the CYP3A4 active site was confirmed by X-ray crystallography. The lead Ir(III)-based probe has nanomolar Kd and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells.

Controlled inhibition of drug-metabolizing cytochrome P450 3A4 (CYP3A4) is utilized to boost bioavailability of anti-viral and immunosuppressant pharmaceuticals. We investigate structure-activity relationships (SARs) in analogues of ritonavir, a potent CYP3A4 inhibitor marketed as pharmacoenhancer, to determine structural elements required for potent inhibition and whether the inhibitory potency can be further improved via a rational structure-based design. This study investigated eight (series VI) inhibitors differing in head- and end-moieties and their respective linkers. SAR analysis revealed the multifactorial regulation of inhibitory strength, with steric constraints imposed on the tethered heme-ligating moiety being a key factor. Minimization of these constraints by changing the linkers' length/flexibility and N-heteroatom position strengthened heme coordination and markedly improved binding and/or inhibitory strength. Impact of the end-pyridine attachment was not uniform due to influence of other determinants controlling the ligand-binding mode. This interplay between pharmacophoric determinants and the end-group enlargement can be used for further inhibitor optimization.

Iron participates as an essential cofactor in the biosynthesis of critical cellular components, including DNA, proteins and lipids. The ergosterol biosynthetic pathway, which is an important target of antifungal treatments, depends on iron in four enzymatic steps. Our results in the model yeast Saccharomyces cerevisiae show that the expression of ergosterol biosynthesis (ERG) genes is tightly modulated by iron availability probably through the iron-dependent variation of sterol and heme levels. Whereas the transcription factors Upc2 and Ecm22 are responsible for the activation of ERG genes upon iron deficiency, the heme-dependent factor Hap1 triggers their Tup1-mediated transcriptional repression. The combined regulation by both activating and repressing regulatory factors allows for the fine-tuning of ERG transcript levels along the progress of iron deficiency, avoiding the accumulation of toxic sterol intermediates and enabling efficient adaptation to rapidly changing conditions. The lack of these regulatory factors leads to changes in the yeast sterol profile upon iron-deficient conditions. Both environmental iron availability and specific regulatory factors should be considered in ergosterol antifungal treatments.

Oxidative stress is defined as an imbalance between oxidants and antioxidants. The reasons behind the condition are diverse, and can result in, or contribute to, the severity of many diseases. The body has different protective mechanisms to reduce the oxidative burden, among them a protein called α1-microglobulin (A1M). A1M possesses a unique set of molecular properties enabling it to act as aradical scavenger, reductase and heme-binder. Most tissues express AMBP, the gene encoding A1M, but the liver is the main site of synthesis. A1M circulates in the blood and is catabolized in the kidneys.Therefore, two interesting sites of action for A1M are the blood and the kidneys.The aim of this thesis was to investigate new therapeutic opportunities for A1M, with focus on renal and erythrocyte pathology. Paper I studied in vitro heme-induced stress in human proximal tubuleepithelial cells. A1M prevented cell death, stress response and mitochondrial dysfunction. In paper II, a 177Lu-DOTATATE cancer treatment mouse model showed that the radiation induced DNA breaks as well as functional and histological damage to the kidneys. The A1M-treated mice, however, showedsignificantly milder damage suggesting that A1M can be used as a radioprotector. Paper III investigated a 177Lu-PSMA-617 prostate cancer treatment mouse model. Only small alterations in kidney function were detected, nonetheless, they were ameliorated in A1M-injected mice. Moreover, it was concluded that A1M does not interfere with treatment.Paper IV presents the establishment and characterization of an A1M-knockout mouse. The lack of A1M generated misfolded bikunin, which is co-expressed with A1M in the intact gene. The A1M-knockout mice also showed increased ER-stress and weight-gain. Paper V shows that the A1Mknockoutmice had fewer red blood cells (RBCs) which also varied more in size, resembling a macrocytic anemia phenotype. Paper V also concludes that A1M has antihemolytic properties, and can protect adult, feta

Mitochondrial carriers (MCs) mediate the passage of small molecules across the inner mitochondrial membrane (IMM), enabling regulated crosstalk between compartmentalized reactions. Despite MCs representing the largest family of solute carriers in mammals, most have not been subjected to a comprehensive investigation, limiting our understanding of their metabolic contributions. Here, we functionally characterize SFXN1, a member of the non-canonical, sideroflexin family. We find that SFXN1, an integral IMM protein with an uneven number of transmembrane domains, is a TIM22 complex substrate. SFXN1 deficiency leads to mitochondrial respiratory chain impairments, most detrimental to complex III (CIII) biogenesis, activity, and assembly, compromising coenzyme Q levels. The CIII dysfunction is independent of one-carbon metabolism, the known primary role for SFXN1 as a mitochondrial serine transporter. Instead, SFXN1 supports CIII function by participating in heme and α-ketoglutarate metabolism. Our findings highlight the multiple ways that SFXN1-based amino acid transport impacts mitochondrial and cellular metabolic efficiency.

Ethnopharmacological relevance: In traditional Chinese medicine (TCM) theory, “Qi” is classified as energetic essence supporting the life activities in human. “Blood” is categorized as nourishing essence and circulating in the body. “Blood” and “Qi” have an intimate relationship. Astragali Radix (AR; root of Astragalus membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao) has a broad spectrum of application for “Qi-Blood” enrichment. Astragaloside IV, a major saponin in AR, has therapeutic functions in erythropoietic, cardiovascular and immune systems. However, the efficacy of astragaloside IV in erythrophagocytosis has not been elucidated. Aim of the study: The possible functions of astragaloside IV in heme iron recycling during erythrophagocytosis in cultured macrophage were elucidated. Methods: The translational and transcriptional expressions of heme recycling enzymes were determined after incubating of astragaloside IV for 24 h in cultured macrophage. Results: In astragaloside IV-treated macrophage, the expressions, both RNA and protein levels, of regulators of heme recycling, e.g. heme oxygenase-1 (HO-1), ferroportin (FPN), biliverdin reductase A and B (BVRA, BVRB), were markedly induced in dose-dependent manners. In parallel, the transcriptional activity of antioxidant response element, cloned within an expression vector as pARE-Luc and transfected in cultured macrophages, was markedly induced after a challenge with astragaloside IV in a dose-dependent manner. Moreover, the translocation of Nrf2, a transcriptional factor in regulating expression of heme recycling protein, was induced by astragaloside IV, leading to an enrichment at nucleus fraction. Conclusion: Astragaloside IV shed lights in enhancing the expression of heme recycle proteins via Nrf2/ARE signaling pathway. © 2020 Elsevier B.V.

The heme enzyme chlorite dismutase (Cld) catalyzes O-O bond formation as part of the conversion of the toxic chlorite (ClO2-) to chloride (Cl-) and molecular oxygen (O2). Enzymatic O-O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae (AoCld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. AoCld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, AoCld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at kobs = 2-5 × 104 s-1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-Timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of AoCld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety's reorganization and thereby maximize the enzyme's catalytic efficiency., BT/Biocatalysis

Heme oxygenase is a cytoprotective enzyme with strong antioxidant and anti-apoptotic properties. Its cytoprotective role is mainly attributed to its enzymatic activity, which involves the degradation of heme to biliverdin with simultaneous release of carbon monoxide (CO). Recent studies uncovered a new cytoprotective role for heme oxygenase-1 (HO-1) by identifying a regulatory role on the complement control protein decay-accelerating factor. This is a key complement regulatory protein preventing dysregulation or overactivation of complement cascades that can cause kidney injury. Cell-specific targeting of HO-1 induction may, therefore, be a novel approach to attenuate complement-dependent forms of kidney disease.

Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. A main feature of DyP is the acidic optimal pH for dye-decolorizing activity. In this study, we constructed several mutant DyP enzymes from Vibrio cholerae (VcDyP), with a view to identifying the decisive factor of the low pH preference of DyP. Initially, distal Asp144, a conserved residue, was replaced with His, which led to significant loss of dye-decolorizing activity. Introduction of His into a position slightly distant from heme resulted in restoration of activity but no shift in optimal pH, indicating that distal residues do not contribute to the pH dependence of catalytic activity. His178, an essential residue for dye decolorization, is located near heme and forms hydrogen bonds with Asp138 and Thr278. While Trp and Tyr mutants of His178 were inactive, the Phe mutant displayed ~35% activity of wild-type VcDyP, indicating that this position is a potential radical transfer route from heme to the active site on the protein surface. The Thr278Val mutant displayed similar enzymatic properties as WT VcDyP, whereas the Asp138Val mutant displayed significantly increased activity at pH 6.5. On the basis of these findings, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity.

Every cell has a highly sophisticated system for regulating heme levels, which is particularly important with regard to turnover. Heme degradation generates CO and while CO has long been viewed as a metabolic waste product, and at higher concentrations cellularly lethal, we now know that CO is an indispensable gasotransmitter that participates in fundamental physiological processes necessary for survival. Irrefutable preclinical data have resulted in concerted efforts to develop CO as a safe and effective therapeutic agent, but against this notion lies dogma that CO is a poison, especially to the brain. The emergence of this debate is discussed here highlighting the neuroprotective properties of CO through its role on the central circadian clock and ongoing strategies being developed for CO administration for clinical use.

Heme proteins have proven to be a convenient platform for the development of designer proteins with novel functionalities. This is achieved by substituting the native iron porphyrin cofactor with a heme analogue that possesses the desired properties. Replacing the iron center of the porphyrin with another metal provides one inroad to novel protein function. A less explored approach is substitution of the porphyrin cofactor with an alternative tetrapyrrole macrocycle or a related ligand. In general, these ligands exhibit chemical properties and reactivity that are distinct from those of porphyrins. While these techniques have most prominently been utilized to develop artificial metalloenzymes, there are many other applications of this methodology to problems in biochemistry, health, and medicine. Incorporation of synthetic cofactors into protein environments represents a facile way to impart water solubility and biocompatibility. It circumvents the laborious synthesis of water-soluble cofactors, which often introduces substantial charge that leads to undesired bioaccumulation. To this end, the incorporation of unnatural cofactors in heme proteins has enabled the development of designer proteins as optical oxygen sensors, MRI contrast agents, spectroscopic probes, tools to interrogate protein function, antibiotics, and fluorescent proteins.Incorporation of an artificial cofactor is frequently accomplished by denaturing the holoprotein with removal of the heme; the refolded apoprotein is then reconstituted with the artificial cofactor. This process often results in substantial protein loss and does not necessarily guarantee that the refolded protein adopts the native structure. To circumvent these issues, our laboratory has pioneered the use of the RP523 strain of E. coli to incorporate artificial cofactors into heme proteins using expression-based methods. This strain lacks the ability to biosynthesize heme, and the bacterial cell wall is permeable to heme and related

Hemoproteins are widely researched because they contain redox-active heme prosthetic groups (iron + protoporphyrin IX) that enable them to perform a range of vital functions, acting as enzymes, participants in electron transfer reactions, or gas sensing, carrying, and storage proteins. While the heme prosthetic group is almost always essential for hemoprotein function, it is frequently desirable to remove it from the protein to enable biochemical or protein engineering studies. Obtaining high yields of the apo form of the hemoprotein can be challenging since high heme-protein binding affinities necessitate the use of harsh conditions to remove heme. In this Bio-Protocol, we present three chemical extraction methods that can be used to efficiently remove heme: methyl ethyl ketone extraction, acid-acetone precipitation, and on-column heme extraction. We also present protocols that can be used to quantitate the amount of residual heme bound to the protein after performing the extraction procedures.

Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. A main feature of DyP is the acidic optimal pH for dye-decolorizing activity. In this study, we constructed several mutant DyP enzymes from Vibrio cholerae (VcDyP), with a view to identifying the decisive factor of the low pH preference of DyP. Initially, distal Asp144, a conserved residue, was replaced with His, which led to significant loss of dye-decolorizing activity. Introduction of His into a position slightly distant from heme resulted in restoration of activity but no shift in optimal pH, indicating that distal residues do not contribute to the pH dependence of catalytic activity. His178, an essential residue for dye decolorization, is located near heme and forms hydrogen bonds with Asp138 and Thr278. While Trp and Tyr mutants of His178 were inactive, the Phe mutant displayed ~35% activity of wild-type VcDyP, indicating that this position is a potential radical transfer route from heme to the active site on the protein surface. The Thr278Val mutant displayed similar enzymatic properties as WT VcDyP, whereas the Asp138Val mutant displayed significantly increased activity at pH 6.5. On the basis of these findings, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity.

Rhabdomyolysis is a life-threatening condition caused by skeletal muscle damage with acute kidney injury being the main complication dramatically worsening the prognosis. Specific treatment for rhabdomyolysis-induced acute kidney injury is lacking and the mechanisms of the injury are unclear. To clarify this, we studied intra-kidney complement activation (C3d and C5b-9 deposits) in tubules and vessels of patients and mice with rhabdomyolysis-induced acute kidney injury. The lectin complement pathway was found to be activated in the kidney, likely via an abnormal pattern of Fut2-dependent cell fucosylation, recognized by the pattern recognition molecule collectin-11 and this proceeded in a C4-independent, bypass manner. Concomitantly, myoglobin-derived heme activated the alternative pathway. Complement deposition and acute kidney injury were attenuated by pre-treatment with the heme scavenger hemopexin. This indicates that complement was activated in a unique double-trigger mechanism, via the alternative and lectin pathways. The direct pathological role of complement was demonstrated by the preservation of kidney function in C3 knockout mice after the induction of rhabdomyolysis. The transcriptomic signature for rhabdomyolysis-induced acute kidney injury included a strong inflammatory and apoptotic component, which were C3/complement-dependent, as they were normalized in C3 knockout mice. The intra-kidney macrophage population expressed a complement-sensitive phenotype, overexpressing CD11b and C5aR1. Thus, our results demonstrate a direct pathological role of heme and complement in rhabdomyolysis-induced acute kidney injury. Hence, heme scavenging and complement inhibition represent promising therapeutic strategies.

Heme oxygenase is a cytoprotective enzyme with strong antioxidant and anti-apoptotic properties. Its cytoprotective role is mainly attributed to its enzymatic activity, which involves the degradation of heme to biliverdin with simultaneous release of carbon monoxide (CO). Recent studies uncovered a new cytoprotective role for heme oxygenase-1 (HO-1) by identifying a regulatory role on the complement control protein decay-accelerating factor. This is a key complement regulatory protein preventing dysregulation or overactivation of complement cascades that can cause kidney injury. Cell-specific targeting of HO-1 induction may, therefore, be a novel approach to attenuate complement-dependent forms of kidney disease.

Biliverdin reductase-A (BVR) catalyzes the reduction of heme-derived biliverdin into bilirubin, this latter being a powerful endogenous free radical scavenger. Furthermore, BVR is also endowed with both serine/threonine/tyrosine kinase and scaffold activities, through which it interacts with the insulin receptor kinase, conventional and atypical protein kinase C isoforms, mitogen-activated protein kinases as well as the phosphatidylinositol-3 kinase/Akt system. By regulating this complex array of signal transduction pathways, BVR is involved in the pathogenesis of neurodegenerative, metabolic, cardiovascular and immune-inflammatory diseases as well as in cancer. In addition, both BVR and BVR-B, this latter being an alternate isozyme predominant during fetal development but sometimes detectable through adulthood, have been studied as peripheral biomarkers for an early detection of Alzheimer's disease, atherosclerosis and some types of cancer. However, despite these interesting lines of evidence, to date BVR has not been considered as an appealing drug target. Only limited evidence supports the neuroprotective effects of atorvastatin and ferulic acid through BVR regulation in the aged canine brain and human neuroblastoma cells, whereas interesting results have been reported regarding the use of BVR-based peptides in preclinical models of cardiac diseases and cancer.

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.

Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels a

Malaria and schistosomiasis are major infectious causes of morbidity and mortality in the tropical and sub-tropical areas. Due to the widespread drug resistance of the parasites, the availability of new efficient and affordable drugs for these endemic pathologies is now a critical public health issue. In this study, we report the design, the synthesis and the preliminary biological evaluation of a series of alkoxyamine derivatives as potential drugs against Plasmodium and Schistosoma parasites. The compounds (RS/SR)-2F, (RR/SS)-2F, and 8F, having IC50 values in nanomolar range against drug-resistant P. falciparum strains, but also five other alkoxyamines, inducing the death of all adult worms of S. mansoni in only 1 h, can be considered as interesting chemical starting points of the series for improvement of the activity, and further structure activity, relationship studies. Moreover, investigation of the mode of action and the rate constants kd for C-ON bond homolysis of new alkoxyamines is reported, showing a possible alkyl radical mediated biological activity. A theoretical chemistry study allowed us to design new structures of alkoxyamines in order to improve the selectivity index of these drugs

Background:Heme and iron homeostasis is perturbed in Alzheimer’s disease (AD); therefore, the aim of the studywas to examine the levels and association of heme with iron-binding plasma proteins in cognitively normal (CN),mild cognitive impairment (MCI), and AD individuals from the Australian Imaging, Biomarker and Lifestyle FlagshipStudy of Ageing (AIBL) and Kerr Anglican Retirement Village Initiative in Ageing Health (KARVIAH) cohorts.Methods:Non-targeted proteomic analysis by high-resolution mass spectrometry was performed to quantify relativeprotein abundances in plasma samples from 144 CN individuals from the AIBL and 94 CN from KARVIAH cohorts and21 MCI and 25 AD from AIBL cohort. ANCOVA models were utilized to assess the differences in plasma proteinsimplicated in heme/iron metabolism, while multiple regression modeling (and partial correlation) was performed toexamine the association between heme and iron proteins, structural neuroimaging, and cognitive measures.Results:Of the plasma proteins implicated in iron and heme metabolism, hemoglobin subunitβ(p= 0.001) was significantlyincreased in AD compared to CN individuals. Multiple regression modeling adjusted for age, sex, APOEε4 genotype, anddisease status in the AIBL cohort revealed lower levels of transferrin but higher levels of hemopexin associated with augmentedbrain amyloid deposition. Meanwhile, transferrin was positively associated with hippocampal volume and MMSE performance,and hemopexin was negatively associated with CDR scores. Partial correlation analysis revealed lack of significant associationsbetween heme/iron proteins in the CN individuals progressing to cognitive impairment.Conclusions:In conclusion, heme and iron dyshomeostasis appears to be a feature of AD. The causal relationship betweenheme/iron metabolism and AD warrants further investigation.

IntroductionCachexia contributes to increased mortality and reduced quality of life in Chronic Obstructive Pulmonary Disease (COPD) and may be associated with underlying gene expression changes. Our goal was to identify differential gene expression signatures associated with COPD cachexia in current and former smokers.MethodsWe analyzed whole-blood gene expression data from participants with COPD in a discovery cohort (COPDGene, N = 400) and assessed replication (ECLIPSE, N = 114). To approximate the consensus definition using available criteria, cachexia was defined as weight-loss > 5% in the past 12 months or low body mass index (BMI) (< 20 kg/m2) and 1/3 criteria: decreased muscle strength (six-minute walk distance < 350 m), anemia (hemoglobin < 12 g/dl), and low fat-free mass index (FFMI) (< 15 kg/m2 among women and < 17 kg/m2 among men) in COPDGene. In ECLIPSE, cachexia was defined as weight-loss > 5% in the past 12 months or low BMI and 3/5 criteria: decreased muscle strength, anorexia, abnormal biochemistry (anemia or high c-reactive protein (> 5 mg/l)), fatigue, and low FFMI. Differential gene expression was assessed between cachectic and non-cachectic subjects, adjusting for age, sex, white blood cell counts, and technical covariates. Gene set enrichment analysis was performed using MSigDB.ResultsThe prevalence of COPD cachexia was 13.7% in COPDGene and 7.9% in ECLIPSE. Fourteen genes were differentially downregulated in cachectic versus non-cachectic COPD patients in COPDGene (FDR < 0.05) and ECLIPSE (FDR < 0.05).DiscussionSeveral replicated genes regulating heme metabolism were downregulated among participants with COPD cachexia. Impaired heme biosynthesis may contribute to cachexia development through free-iron buildup and oxidative tissue damage.

PurposeAcute intermittent porphyria (AIP) is a rare inborn error of heme biosynthesis characterized by life-threatening acute attacks. Few studies have assessed quality of life (QoL) in AIP and those that have had small sample sizes and used tools that may not have captured important domains.MethodsBaseline data from the Porphyrias Consortium's Longitudinal Study were obtained for 259 patients, including detailed disease and medical history data, and the following Patient-Reported Outcomes Measurement Information System (PROMIS) scales: anxiety, depression, pain interference, fatigue, sleep disturbance, physical function, and satisfaction with social roles. Relationships between PROMIS scores and clinical and biochemical AIP features were explored.ResultsPROMIS scores were significantly worse than the general population across all domains, except depression. Each domain discriminated well between asymptomatic and symptomatic patients with symptomatic patients having worse scores. Many important clinical variables like symptom frequency were significantly associated with domain scores in univariate analyses, showing responsiveness of the scales, specifically pain interference and fatigue. However, most regression models only explained ~20% of the variability observed in domain scores.ConclusionPain interference and fatigue were the most responsive scales in measuring QoL in this AIP cohort. Future studies should assess whether these scales capture longitudinal disease progression and treatment response.

To investigate the dehydration associated with protein folding, the partial molar volume changes for protein kunfolding (Delta V-u) in cytochrome c (Cyt c) were determined using high pressure absorption spectroscopy. Delta V-u values for the unfolding to urea- and guanidine hydrochloride (GdnHCl)-denatured Cyt c were estimated to be 56 +/- 5 and 29 +/- 1 mL mol(-1), respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt c has a covalently bonded heme, a positive Delta V-u reflects the primary contribution of the hydration of heme. Because of the marked tendency of guanidium ions to interact with hydrophobic groups, a smaller number of water molecules were hydrated with hydrophobic groups in GdnHCl-denatured Cyt c than in urea-denatured Cyt c, resulting in the smaller positive Delta V-u. On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt c is not completely hydrated, which retains the partially folded structures. To unfold such partial structures, we introduced a mutation near the heme binding site, His26, to Gln, resulting in a negatively shifted Delta V-u (4 +/- 2 mL mol(-1)) in urea- denatured Cyt c. The formation of the more solvated and less structured state in the urea-denatured mutant enhanced hydration to the hydrophilic groups in the unfolding process. Therefore, we confirmed the hydration of amino acid residues in the protein unfolding of Cyt c by estimating Delta V-u, which allows us to discuss the hydrated structures in the denatured states of proteins.

The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacteriumBradyrhizobium japonicum. Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn2+, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr-ICE complex. The addition of EDTA to the Irr-ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn(2+)addition, indicating that Mn(2+)binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn(2+)binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn(2+)release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr-ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29-mutated Irr. These results indicate that Irr requires Mn(2+)binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn(2+)with heme or by heme binding to Cys-29.