Academic literature on the topic 'Functional Modeling - Heme Enzymes'

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.

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.

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.

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.

ABSTRACTTetrapyrroles are ubiquitous molecules in nearly all living organisms. Heme, an iron-containing tetrapyrrole, is widely distributed in nature, including most characterized aerobic and facultative bacteria. A large majority of bacteria that contain heme possess the ability to synthesize it. Despite this capability and the fact that the biosynthetic pathway has been well studied, enzymes catalyzing at least three steps have remained “missing” in many bacteria. In the current work, we have employed comparative genomics via the SEED genomic platform, coupled with experimental verification utilizingAcinetobacter baylyiADP1, to identify one of the missing enzymes, a new protoporphyrinogen oxidase, the penultimate enzyme in heme biosynthesis. COG1981 was identified by genomic analysis as a candidate protein family for the missing enzyme in bacteria that lacked HemG or HemY, two known protoporphyrinogen oxidases. The predicted amino acid sequence of COG1981 is unlike those of the known enzymes HemG and HemY, but in some genomes, the gene encoding it is found neighboring other heme biosynthetic genes. When the COG1981 gene was deleted from the genome ofA. baylyi, a bacterium that lacks bothhemGandhemY, the organism became auxotrophic for heme. Cultures accumulated porphyrin intermediates, and crude cell extracts lacked protoporphyrinogen oxidase activity. The heme auxotrophy was rescued by the presence of a plasmid-borne protoporphyrinogen oxidase gene from a number of different organisms, such ashemGfromEscherichia coli,hemYfromMyxococcus xanthus, or the human gene for protoporphyrinogen oxidase.

Enzymes are proteins that catalyze biochemical reactions and their use report multiple advantages, as they can be very selective, low polluting (biodegradable), cheap and allow working in mild conditions compared with traditional non enzymatic processes. Despite their enormous benefits, their applications at the industrial level are still limited, mainly due to low productivity, low substrate tolerance (too specifics) and poor resistance to the industrial conditions, and for this reason, developing enhanced enzymes by means of enzyme engineering is a central research field nowadays. Notably, the application of computational chemistry in the field of enzyme engineering is increasing due to improvements in hardware and software. Moreover, this process is fast and low-priced and therefore, profitable for the application to the real problems that face industry. Therefore, motivated by this progress, the main goal of this thesis is the development of computational strategies that allow designing and evaluating modifications in enzymes, also aiming to obtain results quickly and inexpensively. This purpose was reached by the combination of different in silico methodologies that were further supported by experimental data in an interactive feedback process. As a result of this thesis, the enzymatic process in heme peroxidases was first satisfactorily described by dividing the process into two steps (from the ligand diffusion to the chemical reaction), using a combination of different computational techniques. The first step, which involves the protein/ligand recognition, was characterized with different molecular mechanics based techniques (MD, Docking and MC-PELE). On the other hand, the chemical reaction (including bond formation and electron transfer) was reproduced using QM based methods by means of energy calculation, spin density characterization, e-coupling calculations and QM/MM e-pathways descriptions. Following this procedure, the oxidation of veratryl alcohol by the enzyme lignin peroxidase was also characterized. Moreover, regarding the e-coupling calculations, a server to compute this vale faster and easy was developed. In the second part of the thesis, the results demonstrated that our protocol could reliably describe and predict enzymatic functions, not only in native enzymes but also in mutated ones, which results were in agreement with experimental data. For example, the structural implications over the reactivity in manganese peroxidase and its engineered variant obtained by cutting the last terminal residues were identified and characterized by the combination of Monte Carlo simulations (PELE) and electronic coupling calculations. The pH resistance in the mutant 2-1B (which was obtained experimentally by random directed evolution) in contrast with the wild type versatile peroxidase, were also rationalized by molecular dynamics, where the residues in the heme environment presented different conformation due to the mutations introduced, resulting in different pH resistance. Interestingly, the last part of the thesis was centered of engineering heme peroxidases. We engineered a peroxidase from in silico predictions to elucidate the long range electron transfer processes involved in the oxidation of the substrate veratryl alcohol by the enzyme versatile peroxidase. In this work we identify the key residues involved in the process, with further applications in engineering enhanced enzymes. Moreover, an enhanced manganese peroxidase mutant from a complete computational study was designed. First, the ligand diffusion study allowed finding the key aminoacids in the substrate/enzyme recognition and binding. Then, the chemical reaction in terms of the oxidation probability and kinetic constant for the proposed mutant were estimated, and the results were in agreement with experimental data. Therefore, the work of this thesis probed that computational biophysics and biochemistry are promising and valuable tools for enzyme engineering. In particular, in the field of rational design of heme peroxidases, they provide relevant information about the enzymatic mechanism and allow designing new enzymes, as well as checking their improvement/worsening, in an efficient way.
Las enzimas son proteínas que catalizan reacciones bioquímicas y cuyo uso aporta múltiples ventajas, ya que son en general muy selectivas, poco contaminantes (biodegradables), baratas y permiten trabajar en condiciones suaves, en comparación con los procesos tradicionales no enzimáticos. A pesar de sus enormes beneficios, sus aplicaciones a nivel industrial son todavía limitadas, debido principalmente a la baja productividad, baja tolerancia al sustrato (demasiado específicos) y una escasa resistencia a las condiciones industriales en general, y por esta razón el desarrollo de enzimas mejoradas es un campo de investigación muy importante hoy en día. En particular, la aplicación de la química computacional en el campo de la ingeniería de enzimas está en aumento debido a las mejoras en hardware y software. Motivado por este progreso, el objetivo principal de esta tesis es el desarrollo de estrategias de cálculo que, mediante la combinación de diferentes metodologías in silico permitan diseñar y evaluar modificaciones en las enzimas, centrándonos en la obtención de resultados de forma rápida y económica. La primera parte de la tesis está centrada en la descripción del mecanismo enzimático entendido como un proceso de dos pasos que incluyen la difusión ligando y la reacción química, mediante una combinación de diferentes técnicas computacionales. El primer paso, que implica el reconocimiento de la proteína / ligando, se caracterizó con diferentes técnicas basadas en la mecánica molecular (dinámica molecular, docking y Monte Carlo- PELE). Por otro lado, la reacción química (incluyendo la formación de enlaces y la transferencia de electrones) se simuló usando métodos basados en mecánica cuántica por medio de cálculos de energía, la caracterización del spin o cálculos de acoplamiento electrónico. Por ejemplo, siguiendo este procedimiento, se caracterizó la oxidación de alcohol veratrílico por medio de la enzima lignin peroxidasa. Además, con el objetivo de poder calcular los acoplamientos electrónicos de una manera más rápida y fácil, se desarrolló un servidor web: ecoupling server. En la segunda parte de la tesis, los resultados demostraron que el protocolo anterior podría describir funciones enzimáticas no sólo en las especies nativas sino también en las variantes mutadas. Por ejemplo, se identificaron las implicaciones estructurales de la reactividad en una manganeso peroxidasa de la subfamilia larga y su variante modificada obtenida mediante la reducción de los últimos residuos terminales gracias al estudio de simulaciones de Monte Carlo (PELE) y cálculos de acoplamiento electrónico. Además, la resistencia a pH ácido en el mutante 2-1B (que se había obtenido previamente por evolución dirigida al azar) se comparó con la especie nativa y también se racionalizó por dinámica molecular, donde se observó que los residuos del entorno del hemo presentaban diferente conformación debido a las mutaciones introducidas, resultando en una diferente resistencia a pH ácido. La última parte de la tesis se centra en la ingeniería racional de hemo peroxidasas. A partir de predicciones in silico se diseñaron variantes de peroxidasa versátil para tratar de entender los procesos de transferencia electrónica de largo alcance que participan en la oxidación del sustrato de alcohol veratrílico, mediante la identificación de los residuos intermedios involucrados en el proceso. Además, a partir de un estudio computacional completo, se diseñó un mutante mejorado de manganeso peroxidasa, cuyos valores cinéticos estimados computacionalmente se encontraban de acuerdo con los resultados experimentales. En conclusión, en esta tesis se ilustra cómo los métodos biofísicos y bioquímicos computacionales son herramientas prometedoras y valiosas para la ingeniería de enzimas, en particular en el campo del diseño racional.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2002.
Includes bibliographical references.
Chapter I. Modeling Dioxygen-Activating Centers in Non-Heme Diiron Enzymes: Carboxylate Shifts in Diiron(II) Complexes Supported by Sterically Hindered Carboxylate Ligands General synthetic routes are described for a series of diiron(II) complexes supported by sterically demanding carboxylate ligands 2,6-di(p-tolyl)benzoate (ArTolCO2-) and 2,6-di(4-fluorophenyl)benzoate (Ar4-FPhCO2-). The interlocking nature of the m-terphenyl units in self-assembled [Fe2(p-O2CArTol)2(O2CArTol)2L2] (L = C5H5N (4); 1-MeIm (5)) promotes the formation of coordination geometries analogous to those of the non-heme diiron cores in the enzymes RNR-R2 and A9D. Magnetic susceptibility and MOssbauer studies of 4 and 5 revealed properties consistent with weak antiferromagnetic coupling between the high-spin iron(II) centers. Structural studies of several derivatives obtained by ligand substitution reactions demonstrated that the [Fe2([mu]O2CAr')2L2] (Ar'=ArTol; Ar 4-FPh) module is geometrically quite flexible. Details of the core rearrangement within the tetracarboxylate diiron framework, facilitated by carboxylate shifts, were probed by solution VT 19F-NMR spectroscopic studies of [Fe2(ap-O2CAr4-FPh)2(O2CAr4-FPh)2(THF)2] (8) and [Fe2(p-O2CAr4-FPh)4(4-tBuC5H4N)2] (12). The dynamic motion in the primary coordination sphere controls the positioning of open sites and regulates the access of exogenous ligands, processes that also occur at the catalytic sites of non-heme diiron enzymes. Chapter II. Structural Flexibility within a Sterically Hindered Ligand Platform: Mononuclear Iron(II) Carboxylate Complexes as Subsite Models for Diiron(II) Centers The synthesis and characterization of a series of mononuclear iron(II) carboxylate complexes are described.
(cont.) By using sterically hindered carboxylate ligands, 2,6-di(p-tolyl)benzoate (ArTolCO2-) and 2,6-di(4-tert-butylphenyl)benzoate (Ar4-tBPhCO2-), series of four-, five-, and six-coordinate iron(II) complexes were synthesized. The compounds are [Fe(O2CArTol)2(1-BnIm)2] (3), [Fe(O2CArTol)2(1-MeBzIm)2] (4), [Fe(02C-Ar4-tBuPh)2(2,2'-bipy)2] (5), [Fe(O2CArTol)2(TMEDA)] (6), and [Fe(O2CArTol)2(BPTA)] (7). Structural analyses of 3-7 revealed that the overall stereochemistry of the [Fe(O2CAr')2Ln] units is dictated by electronic and steric factors of the N-donor ligands (L), as well as by the flexible coordination of the carboxylate ligands. Distinctive MOss-Bauer parameters obtained for these and related compounds facilitated the spectral assignment of a diiron(II) complex having asymmetric metal sites, [Fe2(p-02CArTol)3(2CArTol)(2,6-lutidine)] (2). Well-defined mononuclear iron carboxylate complexes thus may serve as subsite models for higher nuclearity species in both synthetic and biological systems. Chapter III. Functional Mimic of Dioxygen-Activating Centers in Non-Heme Diiron Enzymes: Mechanistic Implications of Paramagnetic Intermediates in the Reactions between Diiron(II) Complexes and Dioxygen Tetracarboxylate diiron(II) complexes, [Fe2(-O02CArTOl)2(02CArToll)2(C5H5N)2] (la) and [Fe2(Pl-02CArTol)4(4-tBuC5H4N)2] (2a), where ArTloCO2- = 2,6-di(p-tolyl)benzoate, react with 02 in CH2C12 at -78 C to afford deep green intermediates ...
by Dongwhan Lee.
Ph.D.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Mononuclear non-heme iron oxygenase (MNO) enzymes utilize ferrous iron and dioxygen to perform a variety of thermodynamically challenging reactions at standard temperatures and pressures. The potent oxidizing power of these enzymatic systems has led to increased interest from the bioinorganic and synthetic organic communities. Presented herein is the preparation and characterization of an α-keto acid dependent synthetic system that closely models the active site electronic and dioxygen reactivity properties of the FeII/α-ketoglutarate dependent class of MNH iron oxygenase enzymes. The ferrous complex utilized possesses a facially coordinating N,N,O-donor ligand reminiscent of a common active site motif observed for MNO iron enzymes. The labile coordination sites opposite the ligand framework allow for the ligation of exogenous α-keto acid cofactor as well as the binding and activation of dioxygen. The coordination of exogenous α-keto acid cofactor has been shown to greatly enhance the rate of dioxygen reactivity of the ferrous complex and lead to the catalytic decarboxylation of the cofactor. The enhancement in rate is attributed to the coupling of the dioxygen reduction step to the oxidative decarboxylation of the bound cofactor, which is a thermodynamically favorable process. The oxidative decarboxylation pathway suggests the formation of a high valent iron-oxo intermediate, which has been further supported by the concentration dependence of solvent oxidation during catalysis. The mechanism of dioxygen reactivity was further probed by Hammett analysis using substituted aromatic α-keto acid cofactors. The data presented suggest that the model system prepared proceeds via a biomimetic mechanism capable of catalytic dioxygen activation and substrate oxidation under ambient conditions. Investigation of differential carboxylate and phenolate ligation as it pertains to MNO iron enzymes is also reported. The synthesis and characterization of both ferrous and ferric compounds containing ligands with similar ethylene diamine backbones and either one or two phenolate moities: 2-(((2-(dimethylarnino)ethyl)(methyl)amino)-methyl)phenol (N2O1-Ph) and 2,2'-((ethane-1,2-diylbis(methylazanediyl))bis-(methylene))diphenol (N2O2-Ph). The replacement of carboxylate moiety with a phenolate led to a significant decrease in reduction potential and subsequent enhancement in dioxygen sensitivity. This observation may provide insight into the reactivity of other iron containing enzymes with coordinated tyrosine residues, such as intradiol catechol dioxygenases.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Vita. Cataloged from student submitted PDF version of thesis.
Includes bibliographical references.
Chapter 1 A comprehensive review of diiron modeling in the Lippard group over the past thirty years is presented. This account describes the different strategies employed to prepare biomimetic complexes of non-heme diiron protein active sites, highlighting the accomplishments of the past as well as the challenges for the future. Studies of various model systems have led to a more profound understanding of the fundamental properties of carboxylate-bridged diiron units and their reactivity toward molecular oxygen and organic substrates. The key principles and lessons that have emerged from these studies have been an inspiration for the original work presented in this thesis. Chapter 2 A series of phenoxylpyridyl and phenoxylimine ligands, H2LR,R' (compounds derived from bis(phenoxylpyridyl)diethynylbenzene, where R = H, Me, or t-Bu, and R' = H, or Ph) and H2BIPSMe,Ph (bis((phenylphenoxyl)iminephenyl)sulfone) were synthesized as platforms for non-heme diiron(II) protein (III) core and molecular oxygen as the source of the bridging oxo group. The [LMe,Ph]2- ligand is robust toward oxidative decomposition and does not display any reversible redox activity. Chapter 3 A dinucleating macrocycle, H2PIM, containing phenoxylimine metal-binding units has been prepared. Reaction of H2PIM with [Fe2(Mes)4] (Mes = 2,4,6-trimethylphenyl) and sterically hindered carboxylic acids, Ph3CCO2H or ArTolCO2H (2,6-bis(p-tolyl)benzoic acid), afforded complexes [Fe2(PIM)(Ph3CCO2)2] (1) and [Fe2(PIM)(ArTolCO2)2] (2), respectively. X-ray diffraction studies revealed that these diiron(II) complexes closely mimic the active site structures of the hydroxylase components of bacterial multi-component monooxygenases (BMMs), particularly the syn disposition of the nitrogen donor atoms and the bridging [mu]--n1n2 and [mu]-n1n1 modes of the carboxylate ligands at the diiron(II) centers. Cyclic voltammograms of 1 and 2 displayed quasi-reversible redox couples at +16 and +108 mV vs. ferrocene/ferrocenium, respectively, assigned to metal-centered oxidations. Treatment of 2 with silver perchlorate afforded a silver(I)/diiron(III) heterotrimetallic complex, [Fe2([mu]-OH)2(CIO4)2(PIM)(ArTolCO2)Ag] (3), which was structurally and spectroscopically characterized. Complexes 1 and 2 both react rapidly with dioxygen. Oxygenation of 1 afforded a ([mu]-hydroxo)diiron(III) complex [Fe2([mu]- OH)(PIM)(Ph3CCO2)3] (4), a hexa([mu]-hydroxo)tetrairon(III) complex [Fe4([mu]- OH)6(PIM)2(Ph3CCO2)2] (5), and an unidentified iron(III) species. Oxygenation of 2 exclusively formed di(carboxylato)diiron(III) products. X-ray crystallographic and 57Fe Mössbauer spectroscopic investigations indicated that 2 reacts with dioxygen to give a mixture of ([mu]- oxo)diiron(III) [Fe2([mu]-O)(PIM)(ArTolCO2)2] (6) and di([mu]-hydroxo)diiron(III) [Fe2([mu]- OH)2(PIM)(ArTolCO2)2] (7) complexes in the same crystal lattice. Compounds 6 and 7 spontaneously convert to a tetrairon(III) complex, [Fe4([mu]-OH)6(PIM)2(ArTolCO2)2] (8), when treated with excess H2O. The possible biological implications of these findings are discussed. Chapter 4 To investigate how protons may be involved in the dioxygen activation pathway of non-heme diiron enzymes, the reaction of H+ with a synthetic ([mu]-1,2-peroxo)(carboxylato)diiron(III) complex was explored. Addition of an H+ donor to [Fe2(O2)(N-EtHPTB)(PhCO2)]2+ (1.O2, where N-EtHPTB = anion of N,N,N' ,N' -tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane) resulted in protonation of the carboxylate rather than the peroxo ligand. Mössbauer and resonance Raman spectroscopic measurements indicate that the Fe2(O2) core of the protonated complex [1.O2]H+ is identical to that of 1.O2. In contrast, the benzoate ligand of [1.O2]H+ displays significantly different IR and NMR spectral features relative to those of the starting complex. The [1.O2]H+ species can be converted back to 1.O2 upon treatment with base, indicating that protonation of the carboxylate is reversible. These findings suggest that in the reaction cycle of soluble methane monooxygenases and related diiron proteins, protons may 6 induce a carboxylate shift to enable substrate access to the diiron core and/or increase the electrophilicity of the oxygenated complex. Chapter 5 To explore additional methods to interrogate the properties of diiron protein intermediates, studies of the vibrational profiles of ([mu]-1,2-peroxo)diiron(III) species were pursued using nuclear resonance vibrational spectroscopy (NRVS). Comparison of the NRVS of [Fe2(O2)(NEtHPTB)(PhCO2)]2+ (1.O2) to that of the diiron(II) starting material [Fe2(N-EtHPTB)(PhCO2)]2+ (1) revealed that the oxygenated complex displays new frequencies above 350 cm-1, which are attributed to the Fe-O-O-Fe core vibrations based on 18O2/16O2 isotopic labeling studies. The peak at 338 cm-1 has not been previously observed by resonance Raman spectroscopy. Empirical normal mode analysis provides a qualitative description of these isotopic sensitive modes. The NRVS of [Fe2([mu]-O2)(HB(iPrpz)3)2(PhCH2CO2)2] (4.O2, where HB(iPrpz)3 = tris(3,5-diisopropylpyrazoyl) hydroborate) was also measured and shows several Fe2(O2) modes between 350-500 cm-1. Appendix A Attempts to prepare a diiron(IV) complex described in the literature led to several unexpected discoveries. Reaction of tris((3,5-dimethyl-4-methoxy)pyridyl-2-methyl)amine (R3TPA) with iron(III) perchlorate decahydrate and sodium hydroxide afforded a ([mu]-oxo)([mu]-hydroxo)diiron(III) [Fe2([mu]-O)([mu]-OH)(R3TPA)2](ClO4)3 complex (1), rather than [Fe2([mu]-O)(OH)(H2O)-(R3TPA)2](ClO4)3 (B) as previously reported. The putative diiron(III) starting material B is formed only at low temperature when excess water is present. Compound 1 hydrolyzes acetonitrile to acetate under ambient conditions. The acetate-bridged diiron compound, [Fe2([mu]- O)([mu]-CH3CO2)(R3TPA)2](ClO4)3 (4A), was characterized by X-ray crystallography as well as various spectroscopic methods and elemental analysis. The identity of the acetate bridged complex was confirmed by comparing the structural and spectroscopic characteristics of 4A to those of an independently prepared sample of [Fe2([mu]-O)([mu]-CH3CO2)(R3TPA)2](ClO4)3.
by Loi Hung Do.
Ph.D.

Dans cette thèse, nous nous sommes intéressés à la relation subtile qui existe entre lesstructures complexes des protéines et leurs fonctions encore plus raffinées que ces dernièreseffectuent. Basés sur deux descriptions différentes des protéines, à l’échelle de acide-aminé età l’echelle atomique, un de nos objectifs était de connecter des indicateurs structuraux calculésà partir de la topologie des protéines à des sites fonctionnels tels que les sites catalyiquesdans les enzymes. Un autre pan de la recherche de cette thèse était d’utiliser nos outils baséssur la structure et de mettre au point de nouvelles simulations numériques pour étudier lesdéterminants basiques structuraux et dynamiques de la communication intramoléculaire dansles protéines. Une première découverte fut de montrer comment l’analyse des modes normauxet la théorie des reseaux complexes conduisent à la prédiction des sites catalytiques dans lesenzymes. De plus, nous avons travaillé sur un groupe relativement peu connu de modes nor-maux qui ont la particularité d’être localisés à deux endroits très eloignés dans la structure desprotéines. Ces modes bilocalisés ont permis de réaliser des transferts d’énergie à des distancesconsidérables (plus de 70 Å). Finalement, des expériences de refroidissement effectuées sur unsystème protéine-eau décrit à l’échelle atomique ont dévoilé que le refroidissement induit unelocalisation spontanée d’énergie, indiquant certaines déformations des anneaux du benzenecomme possible centres de stockage de l’énergie dans les protéines
In this thesis we have focused on the elusive relation that exists in proteins between theircomplex structures and the even more complex and sophisticated functions that they perform.Based on two different descriptions of proteins, at residue and atomistic scale, one of ouraims was to connect structural indicators computed from the topology of protein scaffoldsto hot spots in proteins such as catalytic sites in enzymes. Another goal of this thesis wasto employ our structure-based tools and set up original simulation scheme to investigate thebasic structural and dynamical determinants of intramolecular communication in proteins.As a first important finding, we have shown how normal mode analysis and specific graph-theoretical approaches lead to the prediction of catalytic sites in enzymes. Moreover, wehave concentrated our attention on an overlooked class of normal modes, that are stronglylocalized at two widely separated locations in protein scaffolds. These bilocalized modesturned out to efficiently mediate energy transfer even across considerable distances (morethan 70 Å). Finally, cooling experiments performed on a protein-water system described atatomic level have unveiled complex cooling-induced spontaneous energy localization patterns,pointing to specific deformation modes of benzene rings as potential energy-storage centers

The core of my research activity during the Ph.D. period has been the detection of biological interactions phenomena using electrical transducers, i.e. biosensors. I have studied different aspects of the electrical transduction process, in order to optimize the detection by improving biosensors selectivity and frequency response. I have started my Thesis work by studying the fundamental theories of electrochemical interfaces between biosensor electrodes and liquid samples, e.g., Helmholtz double layer and Warburg frequency dispersion, in order to understand the electron transfer mechanisms in wet environment. The equivalent electrical modeling plays an important role in interpreting experimental electrochemical data. The net flow of electrical charges across an electrochemical interface is the result of several contributions: each of these processes can be modeled using a lumped parameters equivalent electrical circuit with a peculiar electrical impedance. By connecting these equivalent circuits in suitable networks, the frequency response of a complex electrochemical cell can be predicted. During my Ph.D. period I have further developed a simulation system that I started to implement during my Laurea Thesis: with this simulation system the electrical response of an electrode/electrolyte system is predicted using a pseudo-distributed method, i.e. with an interconnection of basic equivalent electrical circuits derived from the geometrical mesh of the simulated system. Each basic equivalent electrical circuit can have different electrical elements and custom topologies. The value of each electrical element, both passive (e.g., resistors and capacitors) and active (e.g., current generators), is determined through mathematical functions elaborated from experimental electrochemical measurements. This mesh-based approach permits to retain the geometrical information of cell and electrodes layout, that is particularly useful when simulating in-flow channel electrodes and microfluidic biosensors. Simulations and equivalent modeling techniques are also useful when designing biosensors layout. During my Ph.D. activity I used commercial biosensors and custom devices: in both cases, the interpretation of experimental data obtained from biosensors with different layouts have been performed by using equivalent electrical circuits modeling techniques, in order to assess the electromagnetic field distribution between electrodes and the influence of parasitics elements, like cross-talk capacitances and tracks intrinsic impedances. During my Ph.D. period I have contributed to develop, in collaboration with Next Step Engineering (University of Padova spin off), an innovative industrial process that allows to create microelectronic/microfluidic hybrid devices within a single, well-established, production line. With this process I have manufactured all the custom devices I used for my experimental activity. Moreover, this industrial process is the object of an Italian patent that is now pending: I have asked for a six months procrastination of my final discussion in order to write and submit the Italian patent for this innovation as one of the inventors. The feasibility of custom biosensors to biomedical and biological applications have been tested using impedance spectroscopy, voltammetric and amperometric measurements: electrical calibration curves have been obtained with standard electrolytes, i.e. solutions with knows electrical conductivity or redox potential, and relevant interferents species have been identified by measuring more complex solutions with various electrolytes and diluted substances. The biological application of custom biosensors have been developed in collaboration with other Departments of the University of Padova and Research Centers: • a genosensor for monitoring DNA hybridization has been developed in collaboration with San Bortolo Hospital (Vicenza, Italy); • an enzyme-modified biosensor for the detection of lactic acid has been studied with the Department of Biomedical Sciences (University of Padova, Italy) and Sapienza University (Roma, Italy); • a biosensor for both monitoring cells growth and studying electropermeabilization has been developed in collaboration with the Department of Biomedical Sciences (University of Padova, Italy). Recently, during the last part of my Ph.D., I studied another application of the electrical transduction of biological signals. In collaboration with Wetware Concepts (University of Padova spin off) and Dr. Quarta from Stanford University, I have contributed to develop a prototype of sensorized glove for the electrical transduction of force signals exerted by human hands. This prototype allows to monitor the functional rehabilitation process of patients with both mild and severe impairments, enabling the quantitative assessment of the functional rehabilitation protocol effectiveness. I have also contributed to further develop the prototype, in collaboration with I.R.C.S.S. San Camillo hospital (Venezia, Italy) and San Bortolo hospital (Vicenza, Italy), into a biofeedback system able to both measure the force exerted by patients hands and to correlate these data with those gathered from other medical equipments, e.g., electroencephalographs and electromyographs.
L’argomento principale dell’attività di ricerca che ho svolto durante il mio periodo di Dottorando in Scienza e Tecnologia dell’Informazione è stato la rilevazione di fenomeni di interazione biologica tramite trasduttori elettrici, ovverosia lo studio di dispositivi elettronici per applicazioni biosensoristiche. Ho studiato diversi aspetti del processo di trasduzione elettrica allo scopo di ottimizzare la rilevazione delle interazioni biologiche e migliorare le caratteristiche dei biosensori, quali ad esempio la selettività e la risposta in frequenza. Ho iniziato il mio lavoro di Tesi studiando le classiche teorie delle interfacce elettrochimiche fra elettrodi metallici e campioni liquidi, ad esempio la teoria del doppio strato di Helmholtz e la dispersione in frequenza di Warburg, per approfondire i meccanismi di trasferimento di carica elettrica in ambienti eterogenei. La modellizzazione elettrica a parametri equivalenti dei dati elettrochimici sperimentali è fondamentale per giungere a una loro interpretazione attendibile: il flusso di cariche elettriche attraverso un’interfaccia elettrochimica è il risultato di numerosi contributi, ciascuno dei quali può essere modellizzato utilizzando circuiti elettrici equivalenti con specifiche impedenze. Collegando questi circuiti equivalenti secondo appropriate topologie è possibile simulare la risposta in frequenza di complesse celle elettrochimiche. Durante il mio periodo di Tesi ho continuato a sviluppare il sistema di simulazione che avevo iniziato a implementare durante il mio periodo di Tesi di Laurea Specialistica: con questo sistema è possibile simulare la risposta elettrica di un sistema elettrodo/elettrolita utilizzando un metodo a elementi pseudo-distribuiti, cioè un’interconnessione finita di circuiti elettrici equivalenti locali la cui topologia viene determinata a partire dalla mesh della geometria della cella elettrochimica. Ciascun circuito elettrico locale può essere formato da diversi elementi elettrici, sia attivi che passivi, con una propria topologia. Il valore di ciascun elemento elettrico locale è determinato con funzioni matematiche ricavate da misure elettrochimiche sperimentali. Questo approccio di simulazione basato sulla mesh consente di preservare le informazioni geometriche legate alla forma degli elettrodi e della cella elettrochimica, che risultano particolarmente importanti quando è necessario simulare elettrodi in flusso oppure biosensori con componenti microfluidiche. Le simulazioni e le tecniche di modellizzazione elettrica risultano importanti anche qualora sia necessario progettare il layout di un biosensore. Durante la mia attività di Dottorato ho utilizzato sia biosensori disponibili commercialmente che dispositivi custom: in entrambi i casi, l’interpretazione dei dati sperimentali ottenuti da biosensori con layout differenti è stata eseguite con tecniche di modellizzazione elettrica equivalente, al fine di valutare la distribuzione del campo elettromagnetico fra gli elettrodi e l’influenza degli elementi parassiti del sistema di misura e del dispositivo, quali ad esempio le capacità di cross-talk e le impedenze elettriche dei contatti elettrici. Durante il mio periodo di Dottorato ho contribuito a sviluppare, in collaborazione con lo spin off dell’Università di Padova Next Step Engineering, un innovativo processo di produzione industriale che consente di creare dispositivi ibridi microelettronici/microfluidici idonei ad applicazioni biologiche all’interno di una singola linea produttiva automatizzata. Con questo processo ho prodotto i dispositivi custom che ho utilizzato per la mia attività sperimentale. Il processo di produzione è oggetto di un brevetto italiano attualmente in fase di deposito, di cui sono uno degli inventori, che ho scritto e sottomesso durante i sei mesi di proroga della discussione finale della Tesi che ho richiesto. La possibilità di utilizzare i biosensori elettrochimici custom per applicazioni biomediche e biologiche è stata verificata utilizzando misurazioni di spettroscopia di impedenza elettrochimica, tecniche voltammetriche e amperometriche: le curve di calibrazione dei vari dispositivi sono state ottenute utilizzando elettroliti standard per le varie applicazioni, cioè soluzioni con conducibilità elettrica e potenziali ossido-riduttivi noti, e l’influenza di interferenti in soluzione è stata valutata misurando matrici più complesse composte da vari elettroliti con sostanze disciolte. Le applicazioni biologiche dei biosensori custom sono state sviluppate in collaborazione con altri Dipartimenti dell’Università degli Studi di Padova e con centri di ricerca: • un biosensore per il monitoraggio dell’ibridazione di sequenze di DNA è stato sviluppato in collaborazione con l’Ospedale San Bortolo (Vicenza, Italia); • un biosensore enzimatico per la rilevazione di acido lattico è stato studiato in collaborazione con il Dipartimento di Scienze Biomediche (Università di Padova, Italia) e con il Dipartimento di Scienze Anatomiche e Istologiche (Università Sapienza, Roma, Italia); • un biosensore per monitorare la crescita cellulare e studiare il fenomeno di elettropermeabilizzazione della membrana cellulare è stato sviluppato in collaborazione con il Dipartimento di Scienze Biomediche (Università di Padova, Italia). Nell’ultimo periodo della mia attività di Dottorato ho studiato un’altra applicazione della trasduzione elettrica di segnali biometrici. In collaborazione con lo spin off dell’Università di Padova Wetware Concepts e con il Dr. Marco Quarta dell’Università di Stanford, ho contribuito a sviluppare un prototipo di guanto sensorizzato per la trasduzione elettrica della forza esercitata da mani umane. Questo prototipo permette di monitorare il processo di riabilitazione funzionale di pazienti con deficit sia lievi che severi, permettendo la valutazione quantitativa dell’efficacia dei protocolli di riabilitazione. Inoltre, ho contribuito a sviluppare ulteriormente il prototipo, in collaborazione con l’I.R.C.C.S. Ospedale San Camillo (Venezia, Italia) e con l’Ospedale San Bortolo (Vicenza, Italia), in un sistema basato su biofeedback in grado di misurare la forza esercitata da un paziente e di correlarla con dati provenienti da altri strumenti medici, quali elettroencefalografi ed elettromiografi.

AbstractCohesive network modelling and systems biology have emerged as extremely potent tools which helps understanding the combinatorial effects of biomolecules. Synergistic modulation among biomolecules (e.g., enzymes, transcription factors, microRNAs, drugs, etc.) are significant in finding out complex regulatory mechanisms in biological networks and pathways. In some cases, although combinatorial interactions among some biomolecules in specific biological networks is available, our knowledge in that particular domain is very limited with context to a genomic scale. Here we explore the pathway-pathway network to identify and understand the network architecture of metabolic pathway mediated regulations at genomic and co-functional levels, in rice. Using network transformation methods, a genome scale pathway-pathway co-functional synergistic network (PcFSN) was constructed. Finally, the PcFSN modules are extracted. This in turn helps to identify the miRNAs and genes associated with the pathways, especially linked to the central metabolic network in rice.

Photodynamic Therapy (PDT) involves administration of a photosensitizer (PS) either systemically or locally, followed by illumination of the lesion with visible light. PDT of cancer is now evolving from experimental treatment to a therapeutic alternative. Clinical results have shown that PDT is at least as efficacious as standard treatments of malignancies of the skin and Barrett’s esophagus. Hemes and heme proteins are vital components of essentially every cell in virtually all eukaryote organisms. Protoporphyrin IX (PpIX) is produced in cells via the heme synthesis pathway from the substrate aminolevulinic acid (ALA). Exogenous administration of ALA induces accumulation of (PpIX), which can be used as a photosensitiser for tumor detection or photodynamic therapy. Although the basis of the selectivity of ALA-based PDT or photodiagnosis is not fully understood, it has sometimes been correlated with the metabolic rate of the cells, or with the differential enzyme expressions along the heme biosynthetic pathway in cancer cells. An in silico analysis by modeling may be performed in order to determine the functional roles of genes coding enzymes of the heme biosynthetic pathway like ferrochelatase. Modeling and simulation systems are a valuable tool for the understanding of complex biological systems. With PyBioS, an object-oriented modelling software for biological processes, we can analyse porphyrin metabolism pathways.

Oxygenases, both flavin-dependent and iron-dependent, act on all classes of natural products, often late in scaffold maturation, to introduce oxygen functional groups, including hydroxyl and epoxide groups. The hydroxyl groups then function as nucleophiles for alkylations, acylations, and glycosylations. The epoxides function as electrophiles in a variety of ring-opening and scaffold rearrangements, as in oxidosqualene cyclizations to sterols. Three variants of iron-containing oxygenases are heme iron (cytochrome P450)-based, nonheme mononuclear iron-based, and di-iron catalysts, all proceeding via high-valent iron-oxo oxidants and radical pathways in cosubstrate oxygen transfers. A substantial fraction of all three types of iron oxygenases act as thwarted oxygenases: the triplet O2 substrate is required to create the high-valent iron-oxo and attendant cosubstrate radical species, but oxygenation half-reactions are not completed. Instead, the cosubstrate radicals react internally and O2 is ultimately reduced to two molecules of H2O. These include penicillin and cephalosporin synthases, three P450s that crosslink the vancomycin heptapeptide backbone, okaramine biogenesis, reticuline to salutaridine in morphine biosynthesis, pinoresinol formation in plant phenylpropanoid pathways, and rebeccamycin and staurosporine indolocarbazole assembly. A third subgrouping of oxygen-consuming enzymes are O2-dependent halogenases. Flavin-dependent halogenases generate HOCl equivalents as sources of [Cl+] ions to electron-rich nucleophiles, while iron-dependent halogenases transfer [Cl<o>˙] equivalents from iron oxychloride complexes, rather than [˙OH] equivalents, to cosubstrate radicals.

The term biological systems may be used in reference to a wide class of polyatomic systems. They can be defined as minimal functional units which perform specific biological functions: enzymatic reactions, transport across membranes, or photosynthesis. At present, such systems as a whole are not amenable to quantum-chemistry studies because of their large size. The smallest enzymes are built of few thousands of atoms (e.g., lysozyme consists of 129 amino-acid subunits), the smallest nucleic acids are of similar size (e.g., t-RNA molecules consist of about 80 nucleotide subunits), whereas biological membranes are even larger and include different biological macromolecules embedded in a phospholipide medium. On the other hand, a common-sense definition of the term biological systems refers to any chemical molecule or molecular complex which is involved in biological or biochemical processes. The latter definition, which will be used throughout this review, covers not only complete functional units performing biological functions but also fragments of such units. Theoretical studies have provided data on properties of such fragments and have helped understanding of the biological processes at the molecular level. Depending upon the size of such fragments, they can be studied by means of various quantum-chemical methods. Molecular systems of up to a few thousands of atoms can be studied using semi-empirical methods. For the Hartree-Fock or Kohn-Sham density functional theory (DFT) calculations, the current size limit is a few hundreds of atoms. (Throughout the text, Hartree-Fock refers to ab initio Self-Consistent Field calculations using the approximation of linear combination of atomic orbitals.) When the desired accuracy requires the calculation of electron correlation at the ab initio level, only systems containing no more than few tens of atoms can be treated. Therefore, a theoretician aiming at the elucidation of biological processes by quantum-mechanical calculations faces two crucial issues. The first one is the selection of a fragment for modeling at the quantum-mechanical level. The second one is the assessment of the effects associated with parts of the system which cannot be modeled at the quantum-mechanical level. In this review, the DFT studies of biological systems are divided into two groups corresponding to different ways of addressing the second aforementioned issue.

Physiological tissue-on-a-chip technology is enabled by adapting microfluidics to create micro scale drug screening platforms that replicate the complex drug transport and reaction processes in the human liver. The ability to incorporate three-dimensional (3d) tissue models using layered fabrication approaches into devices that can be perfused with drugs offer an optimal analog of the in vivo scenario. The dynamic nature of such in vitro metabolism models demands reliable numerical tools to determine the optimum tissue fabrication process, flow, material, and geometric parameters for the most effective metabolic conversion of the perfused drug into the liver microenvironment. Thus, in this modeling-based study, the authors focus on modeling of in vitro 3d microfluidic microanalytical microorgan devices (3MD), where the human liver analog is replicated by 3d cell encapsulated alginate hydrogel based tissue-engineered constructs. These biopolymer constructs are hosted in the chamber of the 3MD device serving as walls of the microfluidic array of channels through which a fluorescent drug substrate is perfused into the microfluidic printed channel walls at a specified volumetric flow rate assuring Stokes flow conditions (Re<<1). Due to the porous nature of the hydrogel walls, a metabolized drug product is collected as an effluent stream at the outlet port. A rigorous modeling approached aimed to capture both the macro and micro scale transport phenomena is presented. Initially, the Stokes Flow Equations (free flow regime) are solved in combination with the Brinkman Equations (porous flow regime) for the laminar velocity profile and wall shear stresses in the whole shear mediated flow regime. These equations are then coupled with the Convection-Diffusion Equation to yield the drug concentration profile by incorporating a reaction term described by the Michael-Menten Kinetics model. This effectively yields a convection-diffusion–cell kinetics model (steady state and transient), where for the prescribed process and material parameters, the drug concentration profile throughout the flow channels can be predicted. A key consideration that is addressed in this paper is the effect of cell mechanotransduction, where shear stresses imposed on the encapsulated cells alter the functional ability of the liver cell enzymes to metabolize the drug. Different cases are presented, where cells are incorporated into the geometric model either as voids that experience wall shear stress (WSS) around their membrane boundaries or as solid materials, with linear elastic properties. As a last step, transient simulations are implemented showing that there exists a tradeoff with respect the drug metabolized effluent product between the shear stresses required and the residence time needed for drug diffusion.