Heme proteins

Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b and non-heme Fe, respectively. The determined sensitivity (-4.00 × 10 ± 1.84 × 10 A/μM and - 2.71 × 10 ± 1.44 × 10 A/μM for NO and O, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O, respecti...

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 is an adaptive and beneficial response in renal and nonrenal settings of tissue injury. The purpose of this study was to characterize the regulation of the human HO-1 gene in renal proximal tubule and aortic endothelial cells in response to heme and cadmium. Evaluation of multiple human HO-1 promoter-reporter constructs up to -9.1 kb demonstrated only a partial response to heme and cadmium. In an effort to mimic endogenous stimulus-dependent levels of HO-1 induction, we evaluated the entire 12.5 kb of the human HO-1 gene, including introns and exons, in conjunction with a -4.5-kb human HO-1 promoter and observed significant heme- and cadmium-mediated induction of the reporter gene, suggesting the presence of an internal enhancer. Enhancer function was orientation independent and required a region between -3.5 and -4.5 kb of the human HO-1 p...

Objective: Heme cofactors are essential molecule found in almost all forms of life. Biological systems depend on heme-protein interactions to carry out basic functions required for their survival. The objective of the present work is to analyse the various non-covalent interactions and also focus on amino acid preferences in heme binding environment. Methods: Various interactions like hydrophobic, aromatic and hydrogen bonds between heme and binding site of non-redundant dataset of 33 heme proteins were analysed to understand the characteristics of different type of interactions. Also the relative preference of amino acids participating in forming secondary structure, solvent accessibility, stabilizing residues and ion-pairs in heme binding environment was analysed. Results: The analysis of heme binding protein environment revealed some important findings, which include the dominant role of non-polar contacts. 12% of the predicted stabilizing residues were also involved in forming interaction with heme. The secondary structure preference analysis showed that 41% of interacting residues preferred to be in helix. The frequency of non-polar amino acids in the buried region was predominant. The preference of amino acid Arg to form complete ion-pair was higher and these ion-pairs formed strong interactions. This provides insights into the better understanding of the heme environment. Conclusion: The present findings through in silico analysis provide valuable information on natural heme binding proteins. These studies will contribute useful information regarding structural stability and its interaction in future designs of novel heme proteins.

SummaryHNOXPred is a webserver for the prediction of gas-sensing heme-nitric oxide/oxygen (H-NOX) proteins from amino acid sequence. H-NOX proteins are gas-sensing hemoproteins found in diverse organisms ranging from bacteria to eukaryotes. Recently, gas-sensing complex multi-functional proteins containing only the conserved amino acids at the heme centers of H-NOX proteins, have been identified through a motif-based approach. Based on experimental data and H-NOX candidates reported in the literature, HNOXPred is created to automate and facilitate the identification of similar H-NOX centers across systems. The server features HNOXSCORES scaled from 0 to 1 that consider in its calculation, the physicochemical properties of amino acids constituting the heme center in H-NOX in addition to the conserved amino acids within the center. From user input amino acid sequence, the server returns positive hits and their calculated HNOXSCORES ordered from high to low confidence which are accompa...

While cytochrome c (Cyt. c) is a soluble protein involved in mitochondrial electron transfer (ET) reactions between Complex III and Complex IV, cytochrome b5 (Cyt. b5) is a microsomal membrane protein acting as a redox aide for diverse cytochrome P450s and their unique reductase. We found little conservation in the sequence and surface amino acid residues of Cyt. c and b5 proteins among evolutionarily diverse species. Hence, the logic that these proteins mediate ET through affinity binding via specific surface residues is weak. Also, analysis of putative protein-protein interactions in the crystal structures of these proteins and their redox partners did not point to any specific interaction logic. The literature on kinetic and thermodynamic constants of mutants (with altered surface residues) did not provide strong evidence to support the binding-based ET paradigm. Topographically divergent Cyt. b5 from one species has been shown to enhance the activity of CYP450 from another speci...

Recently, electron transfers and catalyses in a bevy of redox reactions mediated by hemeproteins were explained by murburn concept. The term "murburn" is abstracted from "" or "" and connotes a novel "" interaction paradigm. Quite unlike the genetic regulations and protein-level affinity-based controls that govern order and specificity/selectivity in conventional treatments, murburn concept is based on stochastic/thermodynamic regulatory principles. The novel insight necessitates a "reactivity outside the active-site" perspective, because select redox enzymatic activity is obligatorily mediated via diffusible radical/species. Herein, reactions employing key hemeproteins (as exemplified by CYP2E1) establish direct experimental connection between "additive-influenced redox catalysis" and "unusual dose responses" in reductionist and physiological milieu. Thus, direct and conclusive molecular-level experimental eviden...

The rise in antibiotic-resistant bacteria caused by the excessive use of antibiotics has led to the urgent exploration of alternative antimicrobial solutions. Among these alternatives, antimicrobial proteins, and peptides (Apps) have garnered attention due to their wide-ranging antimicrobial effects. This study focuses on evaluating the antimicrobial properties of Solanum lycopersicum heme-binding protein 2 (SlHBP2), an apoplastic protein extracted from tomato plants treated with 1-Methyl tryptophan (1-MT), against Pseudomonas syringae pv. tomato DC3000 (Pst). Computational studies indicate that SlHBP2 is annotated as a SOUL heme-binding family protein. Remarkably, recombinant SlHBP2 demonstrated significant efficacy in inhibiting the growth of Pst within a concentration range of 3-25 μg/mL. Moreover, SlHBP2 exhibited potent antimicrobial effects against other microorganisms, including Xanthomonas vesicatoria (Xv), Clavibacter michiganensis subsp. michiganensis (Cmm), and Botrytis cinerea. To understand the mechanism of action employed by SlHBP2 against Pst, various techniques such as microscopy and fluorescence assays were employed. The results revealed that SlHBP2 disrupts the bacterial cell wall and causes leakage of intracellular contents. To summarize, the findings suggest that SlHBP2 has significant antimicrobial properties, making it a potential antimicrobial agent against a wide range of pathogens. Although further studies are warranted to explore the full potential of SlHBP2 and its suitability in various applications.

Pressure is one of the most important parameters controlling the kinetics of chemical reactions. The ability to combine high-pressure techniques with time-resolved spectroscopy has provided a powerful tool in the study of reaction mechanisms. This review is focused on the supporting role of high-pressure kinetic and spectroscopic methods in the exploration of nitric oxide bioinorganic chemistry. Nitric oxide and other reactive nitrogen species (RNS) are important biological mediators involved in both physiological and pathological processes. Understanding molecular mechanisms of their interactions with redox-active metal/non-metal centers in biological targets, such as cofactors, prosthetic groups, and proteins, is crucial for the improved therapy of various diseases. The present review is an attempt to demonstrate how the application of high-pressure kinetic and spectroscopic methods can add additional information, thus enabling the mechanistic interpretation of various NO bioinorg...

Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy, photosynthesis and osmotrophy. Traits associated with the latter two functions arose independently multiple times in the eukaryotes. Fungi successfully coupled osmotrophy with filamentous growth, similar traits are also manifested in the Pseudofungi (oomycetes and hyphochytriomycetes). Both the Fungi and the Pseudofungi encompass a diversity of plant and animal parasites. Genome-sequencing efforts have focused on host-associated microbes (mutualistic symbionts or parasites), providing limited comparisons with free-living relatives. Here we report the first draft genome sequence of a hyphochytriomycete 'pseudofungus'; <i>Hyphochytrium catenoides</i>. Using phylogenomic approaches, we identify genes of recent viral ancestry, with related viral derived genes also present on the genomes of oomycetes, suggesting a complex history of viral coevolution and integration acro...

Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 μmo...

The Os(P)(NO)(ONO) compounds (P = TTP, TMP, OEP, TmTP; TTP = 5,10,15,20-tetra-p-tolylporphyrinato dianion, TMP = 5,10,15,20-tetramesitylporphyrinato dianion, OEP = octaethylporphyrinato dianion, TmTP = tetra(m-tolyl)porphyrinato dianion) have been prepared from the reaction of the precursor carbonyl complexes Os(P)(CO) with excess nitric oxide. Nitrous oxide was detected as a by-product of the reaction. The IR spectra of the Os(P)(NO)(ONO) compounds (as KBr pellets) reveal bands in the 1790–1804 cm–1 range that are assigned to υNO. The IR spectra also reveal two new bands for each complex in the 1495–1531 and 913–962 cm–1 ranges indicative of O-bound nitrito ligands. The linearity of the bound NO groups and the O-binding of the trans nitrito ligands in the Os(P)(NO)(ONO) complexes are evident in the single-crystal X-ray crystal structures of the TTP and TMP derivatives. The kinetics of the reaction were studied by stopped-flow mixing techniques. Spectroscopic analysis of rapidly mix...

This work demonstrates the feasibility of making sensitive nanometer distance measurements between Fe(III) heme centers and nitroxide spin labels in proteins using the double electron-electron resonance (DEER) pulsed EPR technique at 94 GHz. Techniques to measure accurately long distances in many classes of heme proteins using DEER are currently strongly limited by sensitivity. In this paper we demonstrate sensitivity gains of more than 30 times compared with previous lower frequency (X-band) DEER measurements on both human neuroglobin and sperm whale myoglobin. This is achieved by taking advantage of recent instrumental advances, employing wideband excitation techniques based on composite pulses and exploiting more favorable relaxation properties of low-spin Fe(III) in high magnetic fields. This gain in sensitivity potentially allows the DEER technique to be routinely used as a sensitive probe of structure and conformation in the large number of heme and many other metalloproteins.

The division of life into producers and consumers is blurred by evolution. For example, eukaryotic phototrophs can lose the capacity to photosynthesize, although they may retain vestigial plastids that perform other essential cellular functions. Chrysophyte algae have undergone a particularly large number of photosynthesis losses. Here, we present a plastid genome sequence from a nonphotosynthetic chrysophyte, “ Spumella ” sp. NIES-1846, and show that it has retained a nearly identical set of plastid-encoded functions as apicomplexan parasites. Our transcriptomic analysis of 12 different photosynthetic and nonphotosynthetic chrysophyte lineages reveals remarkable convergence in the functions of these nonphotosynthetic plastids, along with informative lineage-specific retentions and losses. At one extreme, Cornospumella fuschlensis retains many photosynthesis-associated proteins, although it appears to have lost the reductive pentose phosphate pathway and most plastid amino acid meta...

Ocular neovascularization underlies major blinding eye diseases such as "wet" age-related macular degeneration (AMD). Despite the successes of treatments targeting the vascular endothelial growth factor (VEGF) pathway, resistant and refractory patient populations necessitate discovery of new therapeutic targets. Using a forward chemical genetic approach, we identified the heme synthesis enzyme ferrochelatase (FECH) as necessary for angiogenesis in vitro and in vivo FECH is overexpressed in wet AMD eyes and murine choroidal neovascularization; siRNA knockdown of Fech or partial loss of enzymatic function in the Fech(m1Pas) mouse model reduces choroidal neovascularization. FECH depletion modulates endothelial nitric oxide synthase function and VEGF receptor 2 levels. FECH is inhibited by the oral antifungal drug griseofulvin, and this compound ameliorates choroidal neovascularization in mice when delivered intravitreally or orally. Thus, FECH inhibition could be used therape...

Recently, electron transfers and catalyses in a bevy of redox reactions mediated by hemeproteins were explained by murburn concept. The term "murburn" is abstracted from "" or "" and connotes a novel "" interaction paradigm. Quite unlike the genetic regulations and protein-level affinity-based controls that govern order and specificity/selectivity in conventional treatments, murburn concept is based on stochastic/thermodynamic regulatory principles. The novel insight necessitates a "reactivity outside the active-site" perspective, because select redox enzymatic activity is obligatorily mediated via diffusible radical/species. Herein, reactions employing key hemeproteins (as exemplified by CYP2E1) establish direct experimental connection between "additive-influenced redox catalysis" and "unusual dose responses" in reductionist and physiological milieu. Thus, direct and conclusive molecular-level experimental eviden...

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 is an adaptive and beneficial response in renal and nonrenal settings of tissue injury. The purpose of this study was to characterize the regulation of the human HO-1 gene in renal proximal tubule and aortic endothelial cells in response to heme and cadmium. Evaluation of multiple human HO-1 promoter-reporter constructs up to -9.1 kb demonstrated only a partial response to heme and cadmium. In an effort to mimic endogenous stimulus-dependent levels of HO-1 induction, we evaluated the entire 12.5 kb of the human HO-1 gene, including introns and exons, in conjunction with a -4.5-kb human HO-1 promoter and observed significant heme- and cadmium-mediated induction of the reporter gene, suggesting the presence of an internal enhancer. Enhancer function was orientation independent and required a region between -3.5 and -4.5 kb of the human HO-1 p...

Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016]. A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equi...

Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vascular tone. Lack of NO bioavailability can result in cardiovascular disease. NO bioavailability is determined by its rates of generation and catabolism; however, it is not known how the NO catabolism rate is regulated in the vascular wall under normoxic, hypoxic, and anaerobic conditions. To investigate NO catabolism under different oxygen concentrations, studies of NO and O2consumption by the isolated rat aorta were performed using electrochemical sensors. Under normoxic conditions, the rate of NO consumption in solution was enhanced in the presence of the rat aorta. Under hypoxic conditions, NO consumption decreased in parallel with the O2concentration. Like the inhibition of mitochondrial respiration by NO, the inhibitory effects of NO on aortic O2consumption increased as O2concentration decreased. Under anaerobic conditions, however, a paradoxical reacceleration of NO consumption occurred. This...