Search Results (22)

Mammalian heme oxygenase (HO) catalyzes heme degradation using reducing equivalents supplied by NADPH–cytochrome P450 reductase (CPR). The tertiary structure of the catalytic domain of a constitutively expressed isoform of HO, HO-2, resembles that of the inductive isoform, HO-1, whereas HO-2 has two heme regulatory motifs (HRM) at the proximal portion of the C-terminus, where the disulfide linkage reflects cellular redox conditions and the second heme binding site is located. Here, we report the results of crosslinking experiments, which suggest that HRM is located near the FMN-binding domain of the CPR when it is complexed with HO-2. The enzymatic assay and reduction kinetics results suggest that heme-bound HRM negatively regulates HO-2 activity in vitro. Cellular redox conditions and free heme concentrations may regulate HO-2 activity. Full article

Mitochondrial holocytochrome c synthase (HCCS) is an essential protein in assembling cytochrome c (cyt c) of the electron transport system. HCCS binds heme and covalently attaches the two vinyls of heme to two cysteine thiols of the cyt c CXXCH motif. Human HCCS recognizes both cyt c and cytochrome c₁ of complex III (cytochrome bc₁). HCCS is mutated in some human diseases and it has been investigated recombinantly by mutational, biochemical, and reconstitution studies in the past decade. Here, we employ structural prediction programs (e.g., AlphaFold 3) on HCCS and its two substrates, heme and cytochrome c. The results, when combined with spectroscopic and functional analyses of HCCS and variants, provide insights into the structural basis for heme binding, apocyt c binding, covalent attachment, and release of the holocyt c product. Results from in vitro reconstitution of purified human HCCS using cyt c and cyt c₁ peptides as acceptors are consistent with the structural modeling of substrate binding. Reconstitution of HCCS and cyt c₁ provides an approach to studying cyt c₁ assembly, which has been refractile to recombinant in vivo reconstitution (unlike HCCS and cyt c). We propose a structural basis for release of the holocyt c product from HCCS based on in vitro studies and on cryoEM structures of the bacterial cyt c synthase (CcsBA) active site. We analyze the kinetoplastid mitochondrial synthase (KCCS), and hypothesize a molecular evolutionary path from mitochondrial endosymbiosis to the current HCCS. Full article

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H₂O₂ detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H₂O₂ substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP. Full article

Recent phylogenetic studies have unveiled a novel class of ascorbate peroxidases called “ascorbate peroxidase-related” (APX-R). These enzymes, found in green photosynthetic eukaryotes, lack the amino acids necessary for ascorbate binding. This study focuses on the sole APX-R from Chlamydomonas reinhardtii referred to as ascorbate peroxidase 2 (APX2). We used immunoblotting to locate APX2 within the chloroplasts and in silico analysis to identify key structural motifs, such as the twin-arginine transport (TAT) motif for lumen translocation and the metal-binding MxxM motif. We also successfully expressed recombinant APX2 in Escherichia coli. Our in vitro results showed that the peroxidase activity of APX2 was detected with guaiacol but not with ascorbate as an electron donor. Furthermore, APX2 can bind both copper and heme, as evidenced by spectroscopic, and fluorescence experiments. These findings suggest a potential interaction between APX2 and plastocyanin, the primary copper-containing enzyme within the thylakoid lumen of the chloroplasts. Predictions from structural models and evidence from ¹H-NMR experiments suggest a potential interaction between APX2 and plastocyanin, emphasizing the influence of APX2 on the copper-binding abilities of plastocyanin. In summary, our results propose a significant role for APX2 as a regulator in copper transfer to plastocyanin. This study sheds light on the unique properties of APX-R enzymes and their potential contributions to the complex processes of photosynthesis in green algae. Full article

Through the degradation of reactive oxygen species (ROS), different antioxidant enzymes, such as catalase (CAT), defend organisms against oxidative stress. These enzymes are crucial to numerous biological functions, like plant development and defense against several biotic and abiotic stresses. However, despite the major economic importance of Avena sativa around the globe, little is known about the CAT gene’s structure and organization in this crop. Thus, a genome-wide investigation of the CAT gene family in oat plants has been carried out to characterize the potential roles of those genes under different stressors. Bioinformatic approaches were used in this study to predict the AvCAT gene’s structure, secondary and tertiary protein structures, physicochemical properties, phylogenetic tree, and expression profiling under diverse developmental and biological conditions. A local Saudi oat variety (AlShinen) was used in this work. Here, ten AvCAT genes that belong to three groups (Groups I–III) were identified. All identified CATs harbor the two conserved domains (pfam00199 and pfam06628), a heme-binding domain, and a catalase activity motif. Moreover, identified AvCAT proteins were located in different compartments in the cell, such as the peroxisome, mitochondrion, and cytoplasm. By analyzing their promoters, different cis-elements were identified as being related to plant development, maturation, and response to different environmental stresses. Gene expression analysis revealed that three different AvCAT genes belonging to three different subgroups showed noticeable modifications in response to various stresses, such as mannitol, salt, and ABA. As far as we know, this is the first report describing the genome-wide analysis of the oat catalase gene family, and these data will help further study the roles of catalase genes during stress responses, leading to crop improvement. Full article

Bacillus subtilis is a model organism for studying Gram-positive bacteria and serves as a cell factory in the industry for enzyme and chemical production. Additionally, it functions as a probiotic in the gastrointestinal tract, modulating the gut microbiota. Its lytic phage SPO1 is also the most studied phage among the genus Okubovrius, including Bacillus phage SPO1 and Camphawk. One of the notable features of SPO1 is the existence of a “host-takeover module”, a cluster of 24 genes which occupies most of the terminal redundancy. Some of the gene products from the module have been characterized, revealing their ability to disrupt host metabolism by inhibiting DNA replication, RNA transcription, cell division, and glycolysis. However, many of the gene products which share limited similarity to known proteins remain under researched. In this study, we highlight the involvement of Gp49, a gene product from the module, in host RNA binding and heme metabolism—no observation has been reported in other phages. Gp49 folds into a structure that does not resemble any protein in the database and has a new putative RNA binding motif. The transcriptome study reveals that Gp49 primarily upregulates host heme synthesis which captures cytosolic iron to facilitate phage development. Full article

In archaea and sulfate-reducing bacteria, heme is synthesized via the siroheme-dependent pathway. The last step of this route is catalyzed by the Radical SAM enzyme AhbD and consists of the conversion of iron-coproporphyrin III into heme. AhbD belongs to the subfamily of Radical SAM enzymes containing a SPASM/Twitch domain carrying either one or two auxiliary iron–sulfur clusters in addition to the characteristic Radical SAM cluster. In previous studies, AhbD was reported to contain one auxiliary [4Fe-4S] cluster. In this study, the amino acid sequence motifs containing conserved cysteine residues in AhbD proteins from different archaea and sulfate-reducing bacteria were reanalyzed. Amino acid sequence alignments and computational structural models of AhbD suggested that a subset of AhbD proteins possesses the full SPASM motif and might contain two auxiliary iron–sulfur clusters (AuxI and AuxII). Therefore, the cluster content of AhbD from Methanosarcina barkeri was studied using enzyme variants lacking individual clusters. The purified enzymes were analyzed using UV/Visible absorption and EPR spectroscopy as well as iron/sulfide determinations showing that AhbD from M. barkeri contains two auxiliary [4Fe-4S] clusters. Heme synthase activity assays suggested that the AuxI cluster might be involved in binding the reaction intermediate and both clusters potentially participate in electron transfer. Full article

Heme is a double-edged sword. On the one hand, it has a pivotal role as a prosthetic group of hemoproteins in many biological processes ranging from oxygen transport and storage to miRNA processing. On the other hand, heme can transiently associate with proteins, thereby regulating biochemical pathways. During hemolysis, excess heme, which is released into the plasma, can bind to proteins and regulate their activity and function. The role of heme in these processes is under-investigated, with one problem being the lack of knowledge concerning recognition mechanisms for the initial association of heme with the target protein and the formation of the resulting complex. A specific heme-binding sequence motif is a prerequisite for such complex formation. Although numerous short signature sequences indicating a particular protein function are known, a comprehensive analysis of the heme-binding motifs (HBMs) which have been identified in proteins, concerning specific patterns and structural peculiarities, is missing. In this report, we focus on the evaluation of known mammalian heme-regulated proteins concerning specific recognition and structural patterns in their HBMs. The Cys-Pro dipeptide motifs are particularly emphasized because of their more frequent occurrence. This analysis presents a comparative insight into the sequence and structural anomalies observed during transient heme binding, and consequently, in the regulation of the relevant protein. Full article

Heme (Fe²⁺-protoporphyrin IX) is a pigment of life, and as a prosthetic group in several hemoproteins, it contributes to diverse critical cellular processes. While its intracellular levels are tightly regulated by networks of heme-binding proteins (HeBPs), labile heme can be hazardous through oxidative processes. In blood plasma, heme is scavenged by hemopexin (HPX), albumin and several other proteins, while it also interacts directly with complement components C1q, C3 and factor I. These direct interactions block the classical pathway (CP) and distort the alternative pathway (AP). Errors or flaws in heme metabolism, causing uncontrolled intracellular oxidative stress, can lead to several severe hematological disorders. Direct interactions of extracellular heme with alternative pathway complement components (APCCs) may be implicated molecularly in diverse conditions at sites of abnormal cell damage and vascular injury. In such disorders, a deregulated AP could be associated with the heme-mediated disruption of the physiological heparan sulphate–CFH coat of stressed cells and the induction of local hemostatic responses. Within this conceptual frame, a computational evaluation of HBMs (heme-binding motifs) aimed to determine how heme interacts with APCCs and whether these interactions are affected by genetic variation within putative HBMs. Combined computational analysis and database mining identified putative HBMs in all of the 16 APCCs examined, with 10 exhibiting disease-associated genetic (SNPs) and/or epigenetic variation (PTMs). Overall, this article indicates that among the pleiotropic roles of heme reviewed, the interactions of heme with APCCs could induce differential AP-mediated hemostasis-driven pathologies in certain individuals. Full article

One of the first clinical observations related to COVID-19 identified hematological dysfunctions. These were explained by theoretical modeling, which predicted that motifs from SARS-CoV-2 structural proteins could bind to porphyrin. At present, there is very little experimental data that could provide reliable information about possible interactions. The surface plasmon resonance (SPR) method and double resonance long period grating (DR LPG) were used to identify the binding of S/N protein and the receptor bind domain (RBD) to hemoglobin (Hb) and myoglobin (Mb). SPR transducers were functionalized with Hb and Mb, while LPG transducers, were only with Hb. Ligands were deposited by the matrix-assisted laser evaporation (MAPLE) method, which guarantees maximum interaction specificity. The experiments carried out showed S/N protein binding to Hb and Mb and RBD binding to Hb. Apart from that, they demonstrated that chemically-inactivated virus-like particles (VLPs) interact with Hb. The binding activity of S/N- and RBD proteins was assessed. It was found that protein binding fully inhibited heme functionality. The registered N protein binding to Hb/Mb is the first experimental fact that supports theoretical predictions. This fact suggests another function of this protein, not only binding RNA. The lower RBD binding activity reveals that other functional groups of S protein participate in the interaction. The high-affinity binding of these proteins to Hb provides an excellent opportunity for assessing the effectiveness of inhibitors targeting S/N proteins. Full article

The mitochondrial large-conductance calcium-activated potassium channel (mitoBK_Ca) is located in the inner mitochondrial membrane and seems to play a crucial role in cytoprotection. The mitoBK_Ca channel is regulated by many modulators, including activators, such as calcium ions and inhibitors, such as heme and its oxidized form hemin. Heme/hemin binds to the heme-binding motif (CXXCH) located between two RCK domains present in the mitochondrial matrix. In the present study, we used the patch-clamp technique in the outside-out configuration to record the activity of mitoBK_Ca channels. This allowed for the application of channel modulators to the intermembrane-space side of the mitoBK_Ca. We found that hemin applied in this configuration inhibits the activity of mitoBK_Ca. In addition, we proved that the observed hemin effect is specific and it is not due to its interaction with the inner mitochondrial membrane. Our data suggest the existence of a new potential heme/hemin binding site in the structure of the mitoBK_Ca channel located on the mitochondrial intermembrane space side, which could constitute a new way for the regulation of mitoBK_Ca channel activity. Full article

Acute kidney injury (AKI) is a major public health concern with significant morbidity and mortality and no current treatments beyond supportive care and dialysis. Preclinical studies have suggested that heme-oxygenase-1 (HO-1), an enzyme that catalyzes the breakdown of heme, has promise as a potential therapeutic target for AKI. Clinical trials involving HO-1 products (biliverdin, carbon monoxide, and iron), however, have not progressed beyond the Phase ½ level. We identified small-molecule inducers of HO-1 that enable us to exploit the full therapeutic potential of HO-1, the combination of its products, and yet-undefined effects of the enzyme system. Through cell-based, high-throughput screens for induction of HO-1 driven by the human HO-1 promoter/enhancer, we identified two novel small molecules and broxaldine (an FDA-approved drug) for further consideration as candidate compounds exhibiting an E_max ≥70% of 5 µM hemin and EC₅₀ <10 µM. RNA sequencing identified shared binding motifs to NRF2, a transcription factor known to regulate antioxidant genes, including HMOX1. In vitro, the cytoprotective function of the candidates was assessed against cisplatin-induced cytotoxicity and apoptosis. In vivo, delivery of a candidate compound induced HO-1 expression in the kidneys of mice. This study serves as the basis for further development of small-molecule HO-1 inducers as preventative or therapeutic interventions for a variety of pathologies, including AKI. Full article

The physiological and biochemical characterization of the “Halloween” genes has fundamental importance in the biosynthesis pathway of ecdysteroids. These genes were found to catalyze the final phases of ecdysteroid biosynthesis from dietary cholesterol to the molting hormone 20-hydroxyecdysone. We report the characterization of the Cs-Phm in a major insect pest in agriculture, the rice striped stem borer, Chilo suppressalis (C. suppressalis). A full-length transcript of Cs-Phm was amplified with an open reading frame (ORF) of 478 amino acids through 5′ and 3′ RACE. Cs-Phm shows five insect-conserved P450 motifs: Helix-C, Helix-I, Helix-K, PERF, and heme-binding motifs. Phylogenetic analysis clearly shows high similarity to Lepidoptera and evolutionary conservation in insects. The relative spatial and temporal transcript profile shows that Cs-Phm is highly expressed in the prothoracic glands and appears throughout the larval development, but with low expression at the start of the larval instar. It seems to peak in 3–4 days and decreases again before the larvae molt. Double-stranded RNA (dsRNA) injection of Cs-Phm at the larval stage efficiently knocked down the target gene and decreased its expression level. The dsRNA-treated group showed significantly decreased ecdysteroid titers, which leads to delayed larval development and higher larval mortality. Negative effects of larval development were rescued by treating 20E in the dsRNA-treated group. Thus, in conclusion, our results suggest that Cs-Phm is functionally conserved in C. suppressalis and encodes functional CYP that contributes to the biogenesis of 20E. Full article

Both the annotation and identification of genes in pathogenic parasites are still challenging. Although, as a survival factor, nitric oxide (NO) has been proven to be synthesized in Trichomonas vaginalis (TV), nitric oxide synthase (NOS) has not yet been annotated in the TV genome. We developed a witness-to-suspect strategy to identify incorrectly annotated genes in TV via the Smith–Waterman and Needleman–Wunsch algorithms through in-depth and repeated alignment of whole coding sequences of TV against thousands of sequences of known proteins from other organisms. A novel NOS of TV (TV NOS), which was annotated as hydrogenase in the NCBI database, was successfully identified; this TV NOS had a high witness-to-suspect ratio and contained all the NOS cofactor-binding motifs (NADPH, tetrahydrobiopterin (BH4), heme and flavin adenine dinucleotide (FAD) motifs). To confirm this identification, we performed in silico modeling of the protein structure and cofactor docking, cloned the gene, expressed and purified the protein, performed mass spectrometry analysis, and ultimately performed an assay to measure enzymatic activity. Our data showed that although the predicted structure of the TV NOS protein was not similar to the structure of NOSs of other species, all cofactor-binding motifs could interact with their ligands with high affinities. We clearly showed that the purified protein had high enzymatic activity for generating NO in vitro. This study provides an innovative approach to identify incorrectly annotated genes in TV and highlights a novel NOS that might serve as a virulence factor of TV. Full article

Extracellular electron transfer (EET), the process that allows microbes to exchange electrons in a redox capacity with solid interfaces such as minerals or electrodes, has been predominantly described in microbes that use iron during respiration. In this work, we characterize the physiology, genome, and electrochemical properties of two obligately heterotrophic marine microbes that were previously isolated from marine sediment cathode enrichments. Phylogenetic analysis of isolate 16S rRNA genes showed two strains, SN11 and FeN1, belonging to the genus Idiomarina. Strain SN11 was found to be nearly identical to I. loihiensis L2-TR^T, and strain FeN1 was most closely related to I. maritima 908087^T. Each strain had a relatively small genome (~2.8–2.9 MB). Phenotypic similarities among FeN1, SN11, and the studied strains include being Gram-negative, motile, catalase- and oxidase-positive, and rod-shaped. Physiologically, all strains appeared to exclusively use amino acids as a primary carbon source for growth. This was consistent with genomic observations. Each strain contained 17 to 22 proteins with heme-binding motifs. None of these were predicted to be extracellular, although seven were of unknown localization and lacked functional annotation beyond cytochrome. Despite the lack of homology to known EET pathways, both FeN1 and SN11 were capable of sustained electron uptake over time in an electrochemical system linked to respiration. Given the association of these Idiomarina strains with electro-active biofilms in the environment and their lack of autotrophic capabilities, we predict that EET is used exclusively for respiration in these microbes. Full article