Journal articles: 'Structural Modeling - Heme Enzymes'

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Jóźwik, Ilona K., Martin Litzenburger, Yogan Khatri, Alexander Schifrin, Marco Girhard, Vlada Urlacher, Andy-Mark W. H. Thunnissen, and Rita Bernhardt. "Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1." Biochemical Journal 475, no. 17 (September 11, 2018): 2801–17. http://dx.doi.org/10.1042/bcj20180402.

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Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium Sorangium cellulosum So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towards ω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B–C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

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Robins, Tiina, Jonas Carlsson, Maria Sunnerhagen, Anna Wedell, and Bengt Persson. "Molecular Model of Human CYP21 Based on Mammalian CYP2C5: Structural Features Correlate with Clinical Severity of Mutations Causing Congenital Adrenal Hyperplasia." Molecular Endocrinology 20, no. 11 (November 1, 2006): 2946–64. http://dx.doi.org/10.1210/me.2006-0172.

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Abstract Enhanced understanding of structure-function relationships of human 21-hydroxylase, CYP21, is required to better understand the molecular causes of congenital adrenal hyperplasia. To this end, a structural model of human CYP21 was calculated based on the crystal structure of rabbit CYP2C5. All but two known allelic variants of missense type, a total of 60 disease-causing mutations and six normal variants, were analyzed using this model. A structural explanation for the corresponding phenotype was found for all but two mutants for which available clinical data are also discrepant with in vitro enzyme activity. Calculations of protein stability of modeled mutants were found to correlate inversely with the corresponding clinical severity. Putative structurally important residues were identified to be involved in heme and substrate binding, redox partner interaction, and enzyme catalysis using docking calculations and analysis of structurally determined homologous cytochrome P450s (CYPs). Functional and structural consequences of seven novel mutations, V139E, C147R, R233G, T295N, L308F, R366C, and M473I, detected in Scandinavian patients with suspected congenital adrenal hyperplasia of different severity, were predicted using molecular modeling. Structural features deduced from the models are in good correlation with clinical severity of CYP21 mutants, which shows the applicability of a modeling approach in assessment of new CYP21 mutations.

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Santana, Margarida, Manuela M. Pereira, Nuno P. Elias, Cláudio M. Soares, and Miguel Teixeira. "Gene Cluster of Rhodothermus marinusHigh-Potential Iron-Sulfur Protein:Oxygen Oxidoreductase, acaa3-Type Oxidase Belonging to the Superfamily of Heme-Copper Oxidases." Journal of Bacteriology 183, no. 2 (January 15, 2001): 687–99. http://dx.doi.org/10.1128/jb.183.2.687-699.2001.

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ABSTRACT The respiratory chain of the thermohalophilic bacteriumRhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream ofrcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E278, Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the CuA domain, that is characteristic ofcaa 3 oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa 3oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa 3 class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.

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Scaffa, Alejandro, George A. Tollefson, Hongwei Yao, Salu Rizal, Joselynn Wallace, Nathalie Oulhen, Jennifer F. Carr, Katy Hegarty, Alper Uzun, and Phyllis A. Dennery. "Identification of Heme Oxygenase-1 as a Putative DNA-Binding Protein." Antioxidants 11, no. 11 (October 28, 2022): 2135. http://dx.doi.org/10.3390/antiox11112135.

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Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in degrading heme into biliverdin and iron. HO-1 can also enter the nucleus and regulate gene transcription independent of its enzymatic activity. Whether HO-1 can alter gene expression through direct binding to target DNA remains unclear. Here, we performed HO-1 CHIP-seq and then employed 3D structural modeling to reveal putative HO-1 DNA binding domains. We identified three probable DNA binding domains on HO-1. Using the Proteinarium, we identified several genes as the most highly connected nodes in the interactome among the HO-1 gene binding targets. We further demonstrated that HO-1 modulates the expression of these key genes using Hmox1 deficient cells. Finally, mutation of four conserved amino acids (E215, I211, E201, and Q27) within HO-1 DNA binding domain 1 significantly increased expression of Gtpbp3 and Eif1 genes that were identified within the top 10 binding hits normalized by gene length predicted to bind this domain. Based on these data, we conclude that HO-1 protein is a putative DNA binding protein, and regulates targeted gene expression. This provides the foundation for developing specific inhibitors or activators targeting HO-1 DNA binding domains to modulate targeted gene expression and corresponding cellular function.

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Timmins, Amy, and Sam P. de Visser. "A Comparative Review on the Catalytic Mechanism of Nonheme Iron Hydroxylases and Halogenases." Catalysts 8, no. 8 (July 31, 2018): 314. http://dx.doi.org/10.3390/catal8080314.

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Enzymatic halogenation and haloperoxidation are unusual processes in biology; however, a range of halogenases and haloperoxidases exist that are able to transfer an aliphatic or aromatic C–H bond into C–Cl/C–Br. Haloperoxidases utilize hydrogen peroxide, and in a reaction with halides (Cl−/Br−), they react to form hypohalides (OCl−/OBr−) that subsequently react with substrate by halide transfer. There are three types of haloperoxidases, namely the iron-heme, nonheme vanadium, and flavin-dependent haloperoxidases that are reviewed here. In addition, there are the nonheme iron halogenases that show structural and functional similarity to the nonheme iron hydroxylases and form an iron(IV)-oxo active species from a reaction of molecular oxygen with α-ketoglutarate on an iron(II) center. They subsequently transfer a halide (Cl−/Br−) to an aliphatic C–H bond. We review the mechanism and function of nonheme iron halogenases and hydroxylases and show recent computational modelling studies of our group on the hectochlorin biosynthesis enzyme and prolyl-4-hydroxylase as examples of nonheme iron halogenases and hydroxylases. These studies have established the catalytic mechanism of these enzymes and show the importance of substrate and oxidant positioning on the stereo-, chemo- and regioselectivity of the reaction that takes place.

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Jortzik, Esther, Kathleen Zocher, Antje Isernhagen, Boniface M. Mailu, Stefan Rahlfs, Giampietro Viola, Sergio Wittlin, Nicholas H. Hunt, Heiko Ihmels, and Katja Becker. "Benzo[b]quinolizinium Derivatives Have a Strong Antimalarial Activity and Inhibit Indoleamine Dioxygenase." Antimicrobial Agents and Chemotherapy 60, no. 1 (October 12, 2015): 115–25. http://dx.doi.org/10.1128/aac.01066-15.

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ABSTRACTThe heme-containing enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) and IDO-2 catalyze the conversion of the essential amino acid tryptophan into kynurenine. Metabolites of the kynurenine pathway and IDO itself are involved in immunity and the pathology of several diseases, having either immunoregulatory or antimicrobial effects. IDO-1 plays a central role in the pathogenesis of cerebral malaria, which is the most severe and often fatal neurological complication of infection withPlasmodium falciparum. Mouse models are usually used to study the underlying pathophysiology. In this study, we screened a natural compound library against mouse IDO-1 and identified 8-aminobenzo[b]quinolizinium (compound 2c) to be an inhibitor of IDO-1 with potency at nanomolar concentrations (50% inhibitory concentration, 164 nM). Twenty-one structurally modified derivatives of compound 2c were synthesized for structure-activity relationship analyses. The compounds were found to be selective for IDO-1 over IDO-2. We therefore compared the roles of prominent amino acids in the catalytic mechanisms of the two isoenzymes via homology modeling, site-directed mutagenesis, and kinetic analyses. Notably, methionine 385 of IDO-2 was identified to interfere with the entrance ofl-tryptophan to the active site of the enzyme, which explains the selectivity of the inhibitors. Most interestingly, several benzo[b]quinolizinium derivatives (6 compounds with 50% effective concentration values between 2.1 and 6.7 nM) were found to be highly effective againstP. falciparum3D7 blood stages in cell culture with a mechanism independent of IDO-1 inhibition. We believe that the class of compounds presented here has unique characteristics; it combines the inhibition of mammalian IDO-1 with strong antiparasitic activity, two features that offer potential for drug development.

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Krone, Nils, Yulia Grischuk, Marina Müller, Ruth Elisabeth Volk, Joachim Grötzinger, Paul-Martin Holterhus, Wolfgang G. Sippell, and Felix G. Riepe. "Analyzing the Functional and Structural Consequences of Two Point Mutations (P94L and A368D) in the CYP11B1 Gene Causing Congenital Adrenal Hyperplasia Resulting from 11-Hydroxylase Deficiency." Journal of Clinical Endocrinology & Metabolism 91, no. 7 (July 1, 2006): 2682–88. http://dx.doi.org/10.1210/jc.2006-0209.

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Abstract Context: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The deficiency of steroid 11-hydroxylase (CYP11B1) resulting from mutations in the CYP11B1 gene is the second most frequent cause. Objective: We studied the functional and structural consequences of two CYP11B1 missense mutations, which were detected in a 1.8-yr-old boy with acne and precocious pseudopuberty, to prove their clinical relevance and study their impact on CYP11B1 function. Results: The in vitro expression studies in COS-7 cells revealed an almost complete absence of CYP11B1 activity for the P94L mutant to 0.05% for the conversion of 11-deoxycortisol to cortisol. The A368D mutant severely reduced the CYP11B1 enzymatic activity to 1.17%. Intracellular localization studies by immunofluorescence revealed that the mutants were correctly localized. Introducing these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the effects measured in vitro. We hypothesize that the A368D mutation interferes with structures important for substrate specificity and heme iron binding, thus explaining its major functional impact. However, according to structural analysis, we would expect only a minor effect of the P94L mutant on 11-hydroxylase activity, which contrasts with the observed major effect of this mutation both in vitro and in vivo. Conclusion: Analyzing the in vitro enzyme function is a complementary procedure to genotyping and a valuable tool for understanding the clinical phenotype of 11-hydroxylase deficiency. This is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides valuable insights in cytochrome P450 structural-functional relationships, although one must be aware of the limitations of in silico-based methods.

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Yadav, Rahul, and Emily E. Scott. "Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes." Journal of Biological Chemistry 293, no. 43 (September 14, 2018): 16623–34. http://dx.doi.org/10.1074/jbc.ra118.005417.

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Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in Escherichia coli growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.

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Afonso, S. G., R. Enriquez de Salamanca, and A. M. Del C. Batlle. "Porphyrin-induced protein structural alterations of heme enzymes." International Journal of Biochemistry & Cell Biology 29, no. 8-9 (August 1997): 1113–21. http://dx.doi.org/10.1016/s1357-2725(97)00045-9.

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Shteinman, A. A. "Structural-functional modeling of non-heme oxygenases." Russian Chemical Bulletin 60, no. 7 (July 2011): 1290–300. http://dx.doi.org/10.1007/s11172-011-0197-5.

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Piontek, K. "Structural biology of ligninolytic enzymes: laccases and heme peroxidases." Acta Crystallographica Section A Foundations of Crystallography 61, a1 (August 23, 2005): c122. http://dx.doi.org/10.1107/s0108767305094857.

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Zuccarello, Lidia, Catarina Barbosa, Smilja Todorovic, and Célia M. Silveira. "Electrocatalysis by Heme Enzymes—Applications in Biosensing." Catalysts 11, no. 2 (February 6, 2021): 218. http://dx.doi.org/10.3390/catal11020218.

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Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability.

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Nemukhin, A. V., B. L. Grigorenko, I. A. Topol, and S. K. Burt. "Modeling dioxygen binding to the non-heme iron-containing enzymes." International Journal of Quantum Chemistry 106, no. 10 (2006): 2184–90. http://dx.doi.org/10.1002/qua.20910.

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Emerson, Joseph P, Erik R Farquhar, and Lawrence Que. "Structural “Snapshots” along Reaction Pathways of Non-Heme Iron Enzymes." Angewandte Chemie International Edition 46, no. 45 (October 8, 2007): 8553–56. http://dx.doi.org/10.1002/anie.200703057.

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Matsunaga, Isamu, and Yoshitsugu Shiro. "Peroxide-utilizing biocatalysts: structural and functional diversity of heme-containing enzymes." Current Opinion in Chemical Biology 8, no. 2 (April 2004): 127–32. http://dx.doi.org/10.1016/j.cbpa.2004.01.001.

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Park, Hyunchang, and Dongwhan Lee. "Ligand Taxonomy for Bioinorganic Modeling of Dioxygen‐Activating Non‐Heme Iron Enzymes." Chemistry – A European Journal 26, no. 27 (March 17, 2020): 5916–26. http://dx.doi.org/10.1002/chem.201904975.

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Wu, Ruiying, Eric Patrick Skaar, Rongguang Zhang, Grazyna Joachimiak, Piotr Gornicki, Olaf Schneewind, and Andrzej Joachimiak. "Staphylococcus aureusIsdG and IsdI, Heme-degrading Enzymes with Structural Similarity to Monooxygenases." Journal of Biological Chemistry 280, no. 4 (October 31, 2004): 2840–46. http://dx.doi.org/10.1074/jbc.m409526200.

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Carrasco, Maria C., and Shabnam Hematian. "(Hydr)oxo-bridged heme complexes: From structure to reactivity." Journal of Porphyrins and Phthalocyanines 23, no. 11n12 (December 2019): 1286–307. http://dx.doi.org/10.1142/s1088424619300258.

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Iron–porphyrins ([Formula: see text] hemes) are present throughout the biosphere and perform a wide range of functions, particularly those that involve complex multiple-electron redox processes. Some common heme enzymes involved in these processes include cytochrome P450, heme/copper oxidase or heme/non-heme diiron nitric oxide reductase. Consequently, the (hydr)oxo-bridged heme species have been studied for the important roles that they play in many life processes or for their application for catalysis and preparation of new functional materials. This review encompasses important synthetic, structural and reactivity aspects of the (hydr)oxo-bridged heme constructs that govern their function and application. The properties and reactivity of the bridging (hydr)oxo moieties are directly dictated by the coordination environment of the heme core, the nature and ligation of the second metal center attached to the (hydr)oxo group, the presence or absence of a linker, and the degree of flexibility around that linker within the scaffold. Here, we summarize the structural features of all known (hydr)oxo-bridged heme constructs and use those to categorize and thus, provide a more comprehensive picture of structure–function relationships.

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Ha, Edward H., Raymond Y. N. Ho, James F. Kisiel, and Joan Selverstone Valentine. "Modeling the Reactivity of .alpha.-Ketoglutarate-Dependent Non-Heme Iron(II)-Containing Enzymes." Inorganic Chemistry 34, no. 9 (April 1995): 2265–66. http://dx.doi.org/10.1021/ic00113a002.

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Zambrano, Gerardo, Emmanuel Ruggiero, Anna Malafronte, Marco Chino, Ornella Maglio, Vincenzo Pavone, Flavia Nastri, and Angela Lombardi. "Artificial Heme Enzymes for the Construction of Gold-Based Biomaterials." International Journal of Molecular Sciences 19, no. 10 (September 24, 2018): 2896. http://dx.doi.org/10.3390/ijms19102896.

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Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.

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Valle-Altamirano, Rodolfo G., Maria Camilla Baratto, Isidro Badillo-Ramírez, Francisco Gasteazoro, Rebecca Pogni, José M. Saniger, and Brenda Valderrama. "Identification of Fe(iii)–OH species as a catalytic intermediate in plant peroxidases at high H₂O₂ concentration." New Journal of Chemistry 46, no. 10 (2022): 4579–86. http://dx.doi.org/10.1039/d1nj04837f.

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The structure for compound III formed after exposure of plant heme peroxidases to excess H2O2 seems to be a hydroxylated form, providing new evidence for understanding the structural basis of the substrate-induced suicidal behavior of these enzymes.

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Mukherjee, Jhumpa, and Sriparna Ray. "Structurally Characterized Non-Heme Fe(IV)Oxo Complexes: A Brief Overview." Asian Journal of Chemistry 34, no. 11 (2022): 2771–85. http://dx.doi.org/10.14233/ajchem.2022.23863.

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Iron(II) centers found in both heme and non-heme enzymes, are the most important metal centre responsible for effectively activating molecular oxygen. Activation of molecular oxygen is important for natural systems and many industrially important reactions. Iron(IV)oxo unit is one of the important intermediates found among the various high valent oxo iron intermediates formed during substrate oxidation in natural enzymes. In this review article, the different synthetic strategies were focused and followed to obtain the X-ray structurally characterized model iron(IV)oxo complexes with non-heme ligands. The ligands were categorized in three different classes and showed how designing a proper ligand, binding with Fe(II) center and reacting it with a suitable oxidizing agent can finally give rise to a system similar to natural systems. Stability of these complexes and some preliminary characterization have also been discussed. The crystallographic characterization of these synthetic models containing iron(IV)oxo intermediates was necessary to understand the mechanistic pathway they follow to mimic the difficult oxidation reactions performed by natural enzymes. In this review, not only the synthetic strategies for these non-heme iron(IV)oxo complexes were highlighted but a detailed structural analysis for these important intermediates were also discussed.

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Todorovic, Smilja, Catarina Barbosa, Lidia Zuccarello, and Celia M. Silveira. "Vibrational Spectro-Electrochemistry of Heme Proteins." ECS Meeting Abstracts MA2022-01, no. 14 (July 7, 2022): 963. http://dx.doi.org/10.1149/ma2022-0114963mtgabs.

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Heme proteins perform a plethora of distinct cellular functions, including e.g. electron transport (ET), energy conversion, detoxification, catalysis, signaling, and gene regulation, and as such inspire a myriad of biotechnological applications. We use resonance Raman (RR) and Surface enhanced RR (SERR) spectroscopies to probe the architecture of the heme pocket in diverse heme proteins and enzymes, which is essential for the understanding of their physiological properties as well as for the evaluation of their potential for development of the 3rd generation bioelectronic devices (1,2). Moreover, plasmonic metal that gives origin to the surface enhancement of the Raman signal of the molecules found in its close proximity can serve as an electrode, thus driving electrochemical processes. In the case of heme proteins attached to plasmonic Ag electrodes, SERR spectra selectively show vibrational bands originating from the heme moiety only, which are sensitive to spin, coordination and redox state and of the heme iron. These properties that govern the catalytic performance of heme enzymes can be monitored in potential dependent manner by SERR spectro-electrochemistry. We have demonstrated that SERR spectro-electrochemistry possesses unique capacity of to i) disentangle ET processes in multi hemic proteins, such as 28 heme containing nitrite reductase, which represent a challenge for all other experimental approaches and ii) detect subtle immobilization induced structural changes in enzymes of biotechnological interest, which e.g. in the case of cytochrome P450 may prevent their successful applications (1-4). Here we show that SERRS monitoring of electrocatalytic processes by immobilized heme peroxidases, can provide information on catalytically relevant species in situ. Several members of a recently discovered family of heme dye-decolorizing peroxidases (DyPs) that possess remarkable catalytic properties in solution and high biotechnological potential, have been immobilized on biocompatible Ag electrodes. Their structural and electrocatalytic properties studied by RR, SERR spectro-electrochemistry and electrochemistry (2). The immobilized DyP from Pseudomonas putida (PpDyP), in particular, shows native structure and outstanding analytical and catalytic parameters, and hence an exceptional potential for development of 3rd generation biosensors for H2O2 detection. In terms of sensitivity, the bioelectrodes carrying immobilized PpDyP outperform HRP based counterparts reported in the literature (2,4). The biosensor based on a PpDyP variant that harbors mutations at the second shell of the heme cavity reveals further improved storage. Our work highlights the importance of integrated, multidisciplinary approach to simultaneously evaluate the structure and catalytic properties of the enzymes, which ensures faster identification and optimization of the promising candidates for biotechnological applications. References: 1. Silveira, C. M.; Moe, E.; Fraaije, M.; Martins, L. O.; Todorovic, S. (2020). Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases. RSC Advances 10, 11095. https://doi.org/10.1039/D0RA00950D 2 Barbosa, C.; Silveira, C. M.; Silva, D.; Brissos, V.; Hildebrandt, P.; Martins, L. O.; Todorovic, S. (2020). Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing. Biosensors and Bioelectronics 153. https://doi.org/10.1016/j.bios.2020.112055 3 Zuccarello, L.; Barbosa, C.; Galdino, E.; Lončar, N.; Silveira, C.M.; Fraaije, M.W.; Todorovic, S. (2021) SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices. Int. J. Mol. Sci. 22, 7998. https://doi.org/10.3390/ijms22157998 4 Zuccarello, L.; Barbosa, C.; Todorovic, S., Silveira, C.M. (2021) Electrocatalysis by Heme Enzymes-Applications in Biosensing. Catalysts 11, 218. https://doi.org/10.3390/catal11020218

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Sugimoto, Hiroshi, Youichi Naoe, Nozomi Nakamura, Akihiro Doi, and Yoshitsugu Shiro. "Inward-facing conformation of the bacterial heme transporter." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1500. http://dx.doi.org/10.1107/s205327331408499x.

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Iron is an essential element for almost all organisms, since iron serves as a catalytic center for redox reactions in many enzymes. Bacterial pathogens need to acquire iron from tissues of host to survive. Heme transport by ATP-binding cassette (ABC) transporter plays a key role in pathological processes. In gram-negative bacteria, the heme or heme protein binds to specific outer membrane receptors on the bacterial surface. The heme is then transported into the cell via ABC transporters. Here, we present the crystal structure of the heme transporter complex BhuUV-T from Burkholderia cenocepacia at 3.5 Å resolution in nucleotide-free state. The permeation pathway created by transmembrane helices of two BhuU subunit exhibits an inward-facing conformation. Comparison with the outward-facing conformation previously reported for the heme transporter HmuUV from Yersinia pestis and homologous vitamin B12 transporter BtuCD-F from E. coli indicates the structural mechanism involving the translational shift of nucleotide binding subunit and repositioning of the helices of permease subunits for substrate translocation. Structure of interface between BhuUV and periplasmic heme-binding protein BhuT suggests that the acidic residues of BhuU at the periplasmic interface may have an important role in releasing the heme from BhuT. We also determined the BhuT in apo and two types of holo form, providing the structural basis for transient and ambiguous heme recognition.

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Mirts, Evan N., Igor D. Petrik, Parisa Hosseinzadeh, Mark J. Nilges, and Yi Lu. "A designed heme-[4Fe-4S] metalloenzyme catalyzes sulfite reduction like the native enzyme." Science 361, no. 6407 (September 13, 2018): 1098–101. http://dx.doi.org/10.1126/science.aat8474.

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Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochromecperoxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.

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Siitonen, Vilja, Brinda Selvaraj, Laila Niiranen, Ylva Lindqvist, Gunter Schneider, and Mikko Metsä-Ketelä. "Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway." Proceedings of the National Academy of Sciences 113, no. 19 (April 25, 2016): 5251–56. http://dx.doi.org/10.1073/pnas.1525034113.

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Nogalamycin, an aromatic polyketide displaying high cytotoxicity, has a unique structure, with one of the carbohydrate units covalently attached to the aglycone via an additional carbon–carbon bond. The underlying chemistry, which implies a particularly challenging reaction requiring activation of an aliphatic carbon atom, has remained enigmatic. Here, we show that the unusual C5′′–C2 carbocyclization is catalyzed by the non-heme iron α-ketoglutarate (α-KG)–dependent SnoK in the biosynthesis of the anthracycline nogalamycin. The data are consistent with a mechanistic proposal whereby the Fe(IV) = O center abstracts the H5′′ atom from the amino sugar of the substrate, with subsequent attack of the aromatic C2 carbon on the radical center. We further show that, in the same metabolic pathway, the homologous SnoN (38% sequence identity) catalyzes an epimerization step at the adjacent C4′′ carbon, most likely via a radical mechanism involving the Fe(IV) = O center. SnoK and SnoN have surprisingly similar active site architectures considering the markedly different chemistries catalyzed by the enzymes. Structural studies reveal that the differences are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. Our findings significantly expand the repertoire of reactions reported for this important protein family and provide an illustrative example of enzyme evolution.

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Li, Huiying, and Thomas L. Poulos. "Structural variation in heme enzymes: a comparative analysis of peroxidase and P450 crystal structures." Structure 2, no. 6 (June 1994): 461–64. http://dx.doi.org/10.1016/s0969-2126(00)00046-0.

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Gumiero, Andrea, Emma J. Murphy, Clive L. Metcalfe, Peter C. E. Moody, and Emma Lloyd Raven. "An analysis of substrate binding interactions in the heme peroxidase enzymes: A structural perspective." Archives of Biochemistry and Biophysics 500, no. 1 (August 2010): 13–20. http://dx.doi.org/10.1016/j.abb.2010.02.015.

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CRISTINO, MARIA DA GLÓRIA G., CARLA CAROLINA F. DE MENESES, MALÚCIA MARQUES SOEIRO, JOÃO ELIAS V. FERREIRA, ANTONIO FLORÊNCIO DE FIGUEIREDO, JARDEL PINTO BARBOSA, RUTH C. O. DE ALMEIDA, JOSÉ C. PINHEIRO, and ANDRÉIA DE LOURDES R. PINHEIRO. "COMPUTATIONAL MODELING OF ANTIMALARIAL 10-SUBSTITUTED DEOXOARTEMISININS." Journal of Theoretical and Computational Chemistry 11, no. 02 (April 2012): 241–63. http://dx.doi.org/10.1142/s0219633612500162.

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Nineteen 10-substitued deoxoartemisinin derivatives and artemisinin with activity against D-6 strains of malarial falciparum designated as Sierra Leone are studied. We use molecular electrostatic potential maps in an attempt to identify key structural features of the artemisinins that are necessary for their activities and molecular docking to investigate the interaction with the molecular receptor (heme). Chemometric modeling: Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA), K-Nearest Neighbor (KNN), Soft Independent Modeling of Class Analogy (SIMCA) and Stepwise Discriminant Analysis (SDA) are employed to reduce dimensionality and investigate which subset of descriptors are responsible for the classification between more active (MA) and less active (LA) artemisinins. The PCA, HCA, KNN, SIMCA and SDA studies showed that the descriptors LUMO (Lowest Unoccupied Molecular Orbital) energy, DFeO1 (Distance between the O 1 atom from ligand and iron atom from heme), X1A (Average Connectivity Index Chi-1) and Mor15u (Molecular Representation of Structure Based on Electron Diffraction) code of signal 15, unweighted, are responsible for separating the artemisinins according to their degree of antimalarial activity. The prediction study was done with a new set of eight artemisinins by using the chemometric methods and five of them were predicted as active against D-6 strains of falciparum malaria. In order to verify if the key structural features that are necessary for their antimalarial activities were investigated for the interaction with the heme, we also carried out calculations of the molecular electrostatic potential (MEP) and molecular docking. MEP maps and molecular docking were analyzed for more active compounds of the prediction set.

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Linde, Dolores, Elena Santillana, Elena Fernández-Fueyo, Alejandro González-Benjumea, Juan Carro, Ana Gutiérrez, Angel T. Martínez, and Antonio Romero. "Structural Characterization of Two Short Unspecific Peroxygenases: Two Different Dimeric Arrangements." Antioxidants 11, no. 5 (April 30, 2022): 891. http://dx.doi.org/10.3390/antiox11050891.

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Unspecific peroxygenases (UPOs) are extracellular fungal enzymes of biotechnological interest as self-sufficient (and more stable) counterparts of cytochrome P450 monooxygenases, the latter being present in most living cells. Expression hosts and structural information are crucial for exploiting UPO diversity (over eight thousand UPO-type genes were identified in sequenced genomes) in target reactions of industrial interest. However, while many thousands of entries in the Protein Data Bank include molecular coordinates of P450 enzymes, only 19 entries correspond to UPO enzymes, and UPO structures from only two species (Agrocybe aegerita and Hypoxylon sp.) have been published to date. In the present study, two UPOs from the basidiomycete Marasmius rotula (rMroUPO) and the ascomycete Collariella virescens (rCviUPO) were crystallized after sequence optimization and Escherichia coli expression as active soluble enzymes. Crystals of rMroUPO and rCviUPO were obtained at sufficiently high resolution (1.45 and 1.95 Å, respectively) and the corresponding structures were solved by molecular replacement. The crystal structures of the two enzymes (and two mutated variants) showed dimeric proteins. Complementary biophysical and molecular biology studies unveiled the diverse structural bases of the dimeric nature of the two enzymes. Intermolecular disulfide bridge and parallel association between two α-helices, among other interactions, were identified at the dimer interfaces. Interestingly, one of the rCviUPO variants incorporated the ability to produce fatty acid diepoxides—reactive compounds with valuable cross-linking capabilities—due to removal of the enzyme C-terminal tail located near the entrance of the heme access channel. In conclusion, different dimeric arrangements could be described in (short) UPO crystal structures.

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Rai, Amrita, Johann P. Klare, Patrick Y. A. Reinke, Felix Englmaier, Jörg Fohrer, Roman Fedorov, Manuel H. Taft, et al. "Structural and Biochemical Characterization of a Dye-Decolorizing Peroxidase from Dictyostelium discoideum." International Journal of Molecular Sciences 22, no. 12 (June 10, 2021): 6265. http://dx.doi.org/10.3390/ijms22126265.

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A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN− molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.

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Fix, Isabelle, Lorenz Heidinger, Thorsten Friedrich, and Gunhild Layer. "The Radical SAM Heme Synthase AhbD from Methanosarcina barkeri Contains Two Auxiliary [4Fe-4S] Clusters." Biomolecules 13, no. 8 (August 18, 2023): 1268. http://dx.doi.org/10.3390/biom13081268.

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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.

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Hagadorn, John R., Lawrence Que, and William B. Tolman. "A Bulky Benzoate Ligand for Modeling the Carboxylate-Rich Active Sites of Non-Heme Diiron Enzymes." Journal of the American Chemical Society 120, no. 51 (December 1998): 13531–32. http://dx.doi.org/10.1021/ja983333t.

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Mahor, Durga, Julia Püschmann, Diederik R. Adema, Marc J. F. Strampraad, and Peter-Leon Hagedoorn. "Unexpected photosensitivity of the well-characterized heme enzyme chlorite dismutase." JBIC Journal of Biological Inorganic Chemistry 25, no. 8 (October 28, 2020): 1129–38. http://dx.doi.org/10.1007/s00775-020-01826-8.

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Abstract Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO2− (chlorite) to innocuous Cl− and O2. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity. Graphic abstract Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between FeIII and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.

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Sugishima, Masakazu, Kei Wada, and Keiichi Fukuyama. "Recent Advances in the Understanding of the Reaction Chemistries of the Heme Catabolizing Enzymes HO and BVR Based on High Resolution Protein Structures." Current Medicinal Chemistry 27, no. 21 (June 15, 2020): 3499–518. http://dx.doi.org/10.2174/0929867326666181217142715.

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In mammals, catabolism of the heme group is indispensable for life. Heme is first cleaved by the enzyme Heme Oxygenase (HO) to the linear tetrapyrrole Biliverdin IXα (BV), and BV is then converted into bilirubin by Biliverdin Reductase (BVR). HO utilizes three Oxygen molecules (O2) and seven electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR) to open the heme ring and BVR reduces BV through the use of NAD(P)H. Structural studies of HOs, including substrate-bound, reaction intermediate-bound, and several specific inhibitor-bound forms, reveal details explaining substrate binding to HO and mechanisms underlying-specific HO reaction progression. Cryo-trapped structures and a time-resolved spectroscopic study examining photolysis of the bond between the distal ligand and heme iron demonstrate how CO, produced during the HO reaction, dissociates from the reaction site with a corresponding conformational change in HO. The complex structure containing HO and CPR provides details of how electrons are transferred to the heme-HO complex. Although the tertiary structure of BVR and its complex with NAD+ was determined more than 10 years ago, the catalytic residues and the reaction mechanism of BVR remain unknown. A recent crystallographic study examining cyanobacterial BVR in complex with NADP+ and substrate BV provided some clarification regarding these issues. Two BV molecules are bound to BVR in a stacked manner, and one BV may assist in the reductive catalysis of the other BV. In this review, recent advances illustrated by biochemical, spectroscopic, and crystallographic studies detailing the chemistry underlying the molecular mechanism of HO and BVR reactions are presented.

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Subedi, Pradeep, Hackwon Do, Jun Hyuck Lee, and Tae-Jin Oh. "Crystal Structure and Biochemical Analysis of a Cytochrome P450 CYP101D5 from Sphingomonas echinoides." International Journal of Molecular Sciences 23, no. 21 (November 1, 2022): 13317. http://dx.doi.org/10.3390/ijms232113317.

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Cytochrome P450 enzymes (CYPs) are heme-containing enzymes that catalyze hydroxylation with a variety of biological molecules. Despite their diverse activity and substrates, the structures of CYPs are limited to a tertiary structure that is similar across all the enzymes. It has been presumed that CYPs overcome substrate selectivity with highly flexible loops and divergent sequences around the substrate entrance region. Here, we report the newly identified CYP101D5 from Sphingomonas echinoides. CYP101D5 catalyzes the hydroxylation of β-ionone and flavonoids, including naringenin and apigenin, and causes the dehydrogenation of α-ionone. A structural investigation and comparison with other CYP101 families indicated that spatial constraints at the substrate-recognition site originate from the B/C loop. Furthermore, charge distribution at the substrate binding site may be important for substrate selectivity and the preference for CYP101D5.

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Guleria, Praveen, and Sudesh Kumar Yadav. "Insights into Steviol Glycoside Biosynthesis Pathway Enzymes Through Structural Homology Modeling." American Journal of Biochemistry and Molecular Biology 3, no. 1 (December 15, 2012): 1–19. http://dx.doi.org/10.3923/ajbmb.2013.1.19.

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Babandi, Abba, Chioma A. Anosike, Lawrence U. S. Ezeanyika, Kemal Yelekçi, and Abdullahi Ibrahim Uba. "Molecular modeling studies of some phytoligands from Ficus sycomorus fraction as potential inhibitors of cytochrome CYP6P3 enzyme of Anopheles coluzzii." Jordan Journal of Pharmaceutical Sciences 15, no. 2 (June 1, 2022): 258–75. http://dx.doi.org/10.35516/jjps.v15i2.324.

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The major obstacle in controlling malaria is the mosquito’s resistance to insecticides, including pyrethroids. The resistance is mainly due to the over-expression of detoxification enzymes such as cytochromes. Insecticides tolerance can be reduced by inhibitors of P450s involved in insecticide detoxification. Here, to design potential CYP6P3 inhibitors, a homology model of the enzyme was constructed using the crystal structure of retinoic acid-bound cyanobacterial CYP120A1 (PDB ID: 2VE3; Resolution: 2.1 Å). Molecular docking study and computational modeling were employed to determine the inhibitory potentials of some phytoligands isolated from Ficus sycomorus against Anopheles coluzzii modeled P450 isoforms, CYP6P3, implicated in resistance. Potential ligand optimization (LE) properties were analyzed using standard mathematical models. Compounds 5, 8,and 9 bound to the Heme iron of CYP6P3 within 3.14, 2.47 and 2.59 Å, respectively. Their respective binding energies were estimated to be -8.93, -10.44, and -12.56 Kcal/mol. To examine the stability of their binding mode, the resulting docking complexes of these compounds with CYP6P3 were subjected to 50 ns MD simulation. The compounds remained bound to the enzyme and Fe (Heme):O (Ligand) distance appeared to be maintained over time. The coordination of a strong ligand to the heme iron shifts the iron from the high- to the stable low-spin form and prevented oxygen from binding to the heme thereby inhibiting the catalytic activity. The LE index showed the high potential of these compounds (5 and 8) to provide a core fragment for optimization into potent P450 inhibitors.

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Stiborová, Marie, Markéta Mikšanová, Václav Martínek, and Eva Frei. "Heme Peroxidases: Structure, Function, Mechanism and Involvement in Activation of Carcinogens. A Review." Collection of Czechoslovak Chemical Communications 65, no. 3 (2000): 297–325. http://dx.doi.org/10.1135/cccc20000297.

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Peroxidases are enzymes playing an important role in large and diverse numbers of physiological processes in organisms including human. We have attempted in this article to summarize and review the important structural and catalytic properties of principal classes of heme peroxidases as well as their biological functions. Major reactions catalyzed by these enzymes (a conventional peroxidase cycle, reactions using O2 and halogenations) and their mechanism are reviewed, too. Moreover, the reaction mechanisms by which peroxidases are implicated in bioactivation of xenobiotic chemicals are presented. Numerous chemicals including protoxicants and procarcinogens are metabolized by equally numerous chemical reactions catalyzed by peroxidases. The unifying theme is the radical nature of the oxidations. The direct conventional peroxidase reaction forming reactive species is generally responsible for the activation of procarcinogenic substrates of peroxidases. The subsequent formation of a superoxide anion radical and peroxy radicals is necessary for activation of chemicals that are poor substrates for peroxidases. The significance of studies concerning the reactions catalyzed by peroxidases is underlined in the present review article. A review with 166 references.

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Heider, Johann, Maciej Szaleniec, Katharina Sünwoldt, and Matthias Boll. "Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation." Journal of Molecular Microbiology and Biotechnology 26, no. 1-3 (2016): 45–62. http://dx.doi.org/10.1159/000441357.

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Ethylbenzene dehydrogenase initiates the anaerobic bacterial degradation of ethylbenzene and propylbenzene. Although the enzyme is currently only known from a few closely related denitrifying bacterial strains affiliated to the Rhodocyclaceae, it clearly marks a universally occurring mechanism used for attacking recalcitrant substrates in the absence of oxygen. Ethylbenzene dehydrogenase belongs to subfamily 2 of the DMSO reductase-type molybdenum enzymes together with paralogous enzymes involved in the oxygen-independent hydroxylation of p-cymene, the isoprenoid side chains of sterols and even possibly n-alkanes; the subfamily also extends to dimethylsulfide dehydrogenases, selenite, chlorate and perchlorate reductases and, most significantly, dissimilatory nitrate reductases. The biochemical, spectroscopic and structural properties of the oxygen-independent hydroxylases among these enzymes are summarized and compared. All of them consist of three subunits, contain a molybdenum-bis-molybdopterin guanine dinucleotide cofactor, five Fe-S clusters and a heme b cofactor of unusual ligation, and are localized in the periplasmic space as soluble enzymes. In the case of ethylbenzene dehydrogenase, it has been determined that the heme b cofactor has a rather high redox potential, which may also be inferred for the paralogous hydroxylases. The known structure of ethylbenzene dehydrogenase allowed the calculation of detailed models of the reaction mechanism based on the density function theory as well as QM-MM (quantum mechanics - molecular mechanics) methods, which yield predictions of mechanistic properties such as kinetic isotope effects that appeared consistent with experimental data.

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Oliveira, Ricardo N. S., Sara R. M. M. de Aguiar, and Sofia R. Pauleta. "Coordination of the N-Terminal Heme in the Non-Classical Peroxidase from Escherichia coli." Molecules 28, no. 12 (June 7, 2023): 4598. http://dx.doi.org/10.3390/molecules28124598.

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The non-classical bacterial peroxidase from Escherichia coli, YhjA, is proposed to deal with peroxidative stress in the periplasm when the bacterium is exposed to anoxic environments, defending it from hydrogen peroxide and allowing it to thrive under those conditions. This enzyme has a predicted transmembrane helix and is proposed to receive electrons from the quinol pool in an electron transfer pathway involving two hemes (NT and E) to accomplish the reduction of hydrogen peroxide in the periplasm at the third heme (P). Compared with classical bacterial peroxidases, these enzymes have an additional N-terminal domain binding the NT heme. In the absence of a structure of this protein, several residues (M82, M125 and H134) were mutated to identify the axial ligand of the NT heme. Spectroscopic data demonstrate differences only between the YhjA and YhjA M125A variant. In the YhjA M125A variant, the NT heme is high-spin with a lower reduction potential than in the wild-type. Thermostability was studied by circular dichroism, demonstrating that YhjA M125A is thermodynamically more unstable than YhjA, with a lower TM (43 °C vs. 50 °C). These data also corroborate the structural model of this enzyme. The axial ligand of the NT heme was validated to be M125, and mutation of this residue was proven to affect the spectroscopic, kinetic, and thermodynamic properties of YhjA.

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42

Franceschi, Lucia De, Mariarita Bertoldi, Maria Domenica Cappellini, Luigia De Falco, Sara Santos Franco, Luisa Ronzoni, Francesco Turrini, Alessandra Colancecco, Clara Camaschella, and Achille Iolascon. "OXIDATIVE STRESS MODULATES HEME LEVELS and INDUCES PEROXIREDOXIN-2 IN β THALASSEMIC ERYTHROPOIESIS as NOVEL CYTOPROTECTIVE RESPONSE." Blood 116, no. 21 (November 19, 2010): 4266. http://dx.doi.org/10.1182/blood.v116.21.4266.4266.

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Abstract Abstract 4266 Beta thalassemia (β-thal) syndromes are worldwide distributed congenital red cell disorders. Increased levels of reactive-oxygen-species (ROS) have been reported to contribute to anemia in β-thal but the mechanism(s) involved in cell protection against ROS damage has only partially investigated. Here, we studied in vitro normal and β-thal erythropoiesis in erythroid cell cultures from CD34+ cells isolated from peripheral blood from adult normal volunteers and from homozygous (bcod39) b-thalassemia patients. We showed increased ROS production in β-thal erythropoiesis and we evaluated the effects of ROS on normal and β-thal erythropoiesis. We carried out a proteomic comparative study, validated by coupling Quantitative-Real time PCR and immunoblot analysis of the differently expressed proteins. We found down-regulation in expression of enzymes involved in heme catabolism such as biliverdin reductase (BVR) and heme-oxygenase-1 (HO-1) and up-regulation of two new cytoprotective cysteine-based-systems: peroxiredoxin-2 (Prx2) and heat-shock-protein-27 (HSP27), while catalase was similarly expressed in both cell models, suggesting a specific pattern of Px2 and HSP27 in β-thal erythroid precursors. We then measured heme levels and during b-thal-erythropoiesis and found that the synthesis of heme was biphasic displaying an increase of heme levels in early phase followed by a decrease in late phase in comparison to controls. Since heme synthesis depends on the erythroid δ-aminolevulinate-synthase isoform (ALAS-2), we evaluated ALAS-2 expression that resulted similar in normal and β-thal erythroid cells. We then showed that ALAS-2 activity was inhibited by both ROS and hemin, suggesting a possible role of heme and ROS levels in regulation of heme biosynthesis in β-thal cells. Since it has been reported that oxidative stress can up-regulate Prx2 expression and that genetically modified cells over-expressing Prx2 are generally more protected from severe oxidative stress (Phalen TJ et al 2006; Rabilloud T et al 2002; Kang SW et al 1998; Zhang P et al 1997), we have hypothesized a cytoprotective role of Prx2 in b-thal-erythropoiesis. We determined that the anti-oxidant Prx2 specifically binds hemin with high and affinity, most likely involving Prx2 cysteine residues. In order to look for the structural determinants to the binding, we noted that both ALAS-2 and Prx2 possess one and two cys-pro motifs, respectively. This motif is generally considered a heme sensor for many proteins able to bind heme and we propose that it could be responsible for heme binding in both enzymes. These data suggest a wider role of Prx2 as both anti-oxidant and heme-binding protein in protective stress-response-systems in β-thal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Chatfield, David C., and Alexander N. Morozov. "Influence of Conserved Structural Elements of the Proximal Pocket in HEME-Thiolate Enzymes on Oxygen Insertion Reactions." Biophysical Journal 114, no. 3 (February 2018): 585a. http://dx.doi.org/10.1016/j.bpj.2017.11.3202.

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Andersson, Laura A., Anna K. Johnson, Melissa D. Simms, and Timothy R. Willingham. "Comparative analysis of catalases: spectral evidence against heme-bound water for the solution enzymes." FEBS Letters 370, no. 1-2 (August 14, 1995): 97–100. http://dx.doi.org/10.1016/0014-5793(95)00651-o.

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Majumdar, Amit, and Sabyasachi Sarkar. "Bioinorganic chemistry of molybdenum and tungsten enzymes: A structural–functional modeling approach." Coordination Chemistry Reviews 255, no. 9-10 (May 2011): 1039–54. http://dx.doi.org/10.1016/j.ccr.2010.11.027.

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Ziemys, A., and J. Kulys. "Heme peroxidase clothing and inhibition with polyphenolic substances revealed by molecular modeling." Computational Biology and Chemistry 29, no. 2 (April 2005): 83–90. http://dx.doi.org/10.1016/j.compbiolchem.2004.12.007.

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Abraham, Nader G., Jean-Michel Camadro, Sylvia T. Hoffstein, and Richard D. Levere. "Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver." Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 870, no. 2 (March 1986): 339–49. http://dx.doi.org/10.1016/0167-4838(86)90238-4.

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Nóbrega, Cláudia S., Ana Luísa Carvalho, Maria João Romão, and Sofia R. Pauleta. "Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase—Insights into the Catalytic Cycle of Bacterial Peroxidases." International Journal of Molecular Sciences 24, no. 7 (March 26, 2023): 6246. http://dx.doi.org/10.3390/ijms24076246.

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Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.

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Wan, Dun, Li Fu Liao, and Ying Wu Lin. "Impacts of Uranyl Ion on the Structure and Function of Cytochrome b₅ His39Ser Mutant." Advanced Materials Research 455-456 (January 2012): 1204–9. http://dx.doi.org/10.4028/www.scientific.net/amr.455-456.1204.

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Uranium is toxic to human body with mechanisms not fully understood. The structural and functional consequences of uranyl ions (UO22+) interacting with an axial mutant of cytochrome b5, His39Ser (cyt b5 H39S), were investigated by both spectroscopic and molecular modeling methods. Although slightly disturbs protein folding, UO22+ binding to cyt b5 H39S leads to a decrease of peroxidase activity. A uranyl binding site was further proposed in the heme-binding domain at Glu37 and Glu43. The impacts of UO22+ binding to cyt b5 H39S studied herein provide valuable insights into the toxicity mechanism of UO22+ towards membrane heme proteins.

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Hegg, Eric L., and Lawrence Que Jr. "The 2-His-1-Carboxylate Facial Triad - An Emerging Structural Motif in Mononuclear Non-Heme Iron(II) Enzymes." European Journal of Biochemistry 250, no. 3 (December 1997): 625–29. http://dx.doi.org/10.1111/j.1432-1033.1997.t01-1-00625.x.

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Journal articles on the topic 'Structural Modeling - Heme Enzymes'

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