Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN- complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.

The productive, controlled activation of O2 via small-molecule catalysts remains a significant challenge to bioinorganic chemistry. With enzymes, global folding energies dictate the positioning of ligands to optimize chemical pathways. Synthetic iron catalytic complexes, with open or labile coordination sites, frequently give rise to Fenton-like radical-based reactions. To minimize such peroxide/hydroxyl radicals, we developed a synthetic model for the 2-oxoglutarate-dependent dioxygenases, [FeII(N2O1)]- (N2O1 = 2-((2-dimethylamino)ethyl)-(methyl)amino)acetic acid) . We present herein an iron-based synthetic analogue system capable of utilizing dioxygen to perform velut vivum C ‒ H activation at ambient temperatures and pressures. The relationships between a range of α-ketocarboxylate adducts producing metal-to-ligand charge transfer band energies and their corresponding π → π* energy gaps (DFT simulated) are described. Electronic (MCD) and computational (DFT) methods are combined to elucidate the electronic structures of a subset of these adducts. Catalytic efficiency of 15 α-ketocarboxylates is investigated and includes the use of oxalate. The latter represents an original example of a small-molecule synthetic catalyst capable of activating O2 while using oxalate as a cofactor for C ‒ H oxidation. A series of varying N,N,Ox (x = 1 ̶ 3) carboxylate-ligated ferrous, ferric, and chromic complexes was assessed for chemical and electronic influences of increasing carboxylate metal ligation. Electrochemical and spectroscopic characterization, and initial reactivities were examined. The use of the same ligand set but with differing ‘d-electron count’ explores mechanistically dramatic changes to chemical reactivity in the presence of a terminal oxidant. A methodology for the functionalization of the N2Ox (x = 1 ̶ 3) ligand series, using an alkynyl moiety, was also developed. Such could allow ‘click’ chemistry for converting homogeneous catalysts to heterogeneous versions. Using a globally uniform and diffuse low loading resin will provide enhanced catalyst lifetime by diminishing inactivation pathways and support its use in flow chemistry reactors using O2 from air as the oxidant.

Tin protoporphyrin IX (SnPP) is currently under investigation for the treatment of hyperbilirubinemia. The study of the complex between SnPP and equine myoglobin (EqMb) by ¹H and ¹¹⁹Sn nuclear magnetic resonance spectroscopy (NMR) can be viewed as a general model for SnPP interaction with hemoproteins. The complex formed from the equilibrium mixture of SnPP and EqMb, SnPP•EqMb, was found to have essentially the same porphyrin-binding pocket as EqMbCO and SwMbCO, including the same porphyrin orientation in the major form of the two species. ¹¹⁹Sn NMR spectroscopy was used to demonstrate that the proximal His(93)F8-metal coordination is likely to be intact in SnPP•EqMb. Minor shifts in the side chain positions of some of the residues were observed, possibly reflecting the presence of water in the sixth coordination site. SnPP•EqMb appears to be stable; it persists at room temperature for weeks and exhibits very slow exchange rates (²Hfor ¹H) for a large number of amide protons in the pH range 7-9. Events during the reconstitution of apomyoglobin (apoMb) with SnPP were probed. Thus interactions between tin(IV)protoporphyrin IX (SnPP) and equine apoMb, and between tin(IV) protoporphyrin IX dimers (SnPP)₂ and apoMb were observed by ¹H NMR and optical spectroscopic techniques. The products and intermediates observed in this situation were related to the equilibrium structure of SnPP•EqMb. Reactions of apoEqMb with SnPP and (SnPP)₂ produce different intermediates, although the final product, SnPP•EqMb, is the same for each. An intermediate observed for the reaction of SnPP with apoEqMb at pH 10 is in exchange with free SnPP, with the observed rate constant Koff ~ 1 sˉ¹; meso-proton resonances were assigned for this intermediate by correlation to SnPP resonances via chemical exchange. The intermediate observed for the reaction of (SnPP)₂ with apoEqMb at neutral pH produces another species which may be the alternate porphyrin-insertion isomer arising from a 180° rotation about the α,γ-meso axis of the porphyrin. Although optical absorbance spectroscopy of the Soret region shows evidence for the reaction of SnPP and (SnPP)₂ with apoMb, only in combination with ¹H NMR are the various processes assigned. T his study of the complex SnPP•EqMb facilitated the investigation of the more complex heme binding protein, hemopexin (Hx). Proton NMR spectroscopy is reported for the first time for the hemin complex of hemopexin, a serum protein that binds heme exceptionally tightly. Hx from cow, rat, rabbit, and human was isolated, and data for the protein were reported. Heme-bound Hx has spectral characteristics for being low-spin, paramagnetic. Deuterium isotope labels reveal the positions for the heme 1-, 3-, and 8-methyls; the 5-methyl lies in the -5 to 12 ppm region. Furthermore, two-dimensional nuclear Overhauser effect spectroscopy was used to locate other heme periphery protons, including those from the 2-vinyl and the 7-propionate. Upfield resonances are identified that are very strongly relaxed, and so are assigned to protons on the axial ligands. The information reported here contributes to the understanding of Hx as an antioxidant at the cellular level.

G-quadruplexes, a type of nucleic acid secondary structure consisting largely of folded quartets of guanines, appear to play a regulatory role in the human genome. Heme has been shown to interact with G-quadruplexes. The ChIP-Seq-like Heme-Seq assay was developed to identify heme binding G-quadruplex loci. Using Heme-Seq, 3 primary heme binding loci and 4 secondary minor heme binding loci were identified on six chromosomes. Two of the primary heme binding loci were found at the centromeric boundaries of the long arms of metacentric chromosomes with the majority of reads from the primary heme binding loci consisting primarly of Human Satellite II (HSATII) nucleotide repeat sequences. Numerous putative G-quadruplex forming sequences were found in the heme-binding locus on Chromosome 2. Comparison of Heme-Seq results with available data from a G-quadruplex ChIP-Seq study in live cells, revealed that the regions which exhibited binding at the three peaks from the Heme-Seq data also showed binding coverage in the CHIP-Seq data. In addition to the known association with G-quadruplexes, heme also appears to bind to HSATII repeats,. The biological role and importance of this binding is not known. / Biomedical Sciences

No description available.

The Gram-positive pathogens Corynebacterium diphtheriae and Streptococcus pyogenes both require iron for survival. These bacteria have developed sophisticated heme uptake and transport protein machinery responsible for the import of iron into the cell, in the form of heme from the human host. The heme utilization pathway (hmu) of C. diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. The axial ligation of the heme in HmuT was probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, R237A, Y272A, M292A, Y349A, and Y349F. Characterization by UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopies indicated that H136 and Y235 are the axial ligands in HmuT. Electrospray ionization mass spectrometry was also utilized to assess the roles of conserved residues in contribution to heme binding. The S. pyogenes streptococcal iron acquisition (sia)/heme transport system (hts) utilizes multiple proteins to bring host heme to the intracellular space. Both the substrate binding protein SiaA and the hemoprotein surface receptor Shr were investigated. The kinetic effects on SiaA heme release were probed through chemical unfolding of axial ligand mutants M79A and H229A, as well mutants thought to contribute to heme binding, K61A and C58A, and a control mutant, C47A. The unfolding pathways showed two processes for protein denaturation. This is consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket. The ease of protein unfolding is related to the strength of interaction of the residues with the heme. Shr contains two NEAT (near-iron transporter) domains (Shr-N1 and Shr-N2) which can both bind heme. Biophysical studies of both Shr-N1 and Shr-N2 indicated a new class of NEAT domains which utilize methionine as an axial ligand, rather than a tyrosine. Thermal and chemical unfolding showed ferrous Shr-N1 and Shr-N2 to be most resistant to denaturation. Shr-N2 was prone to autoreduction. Together, sequence alignment, homology modeling, and spectral signatures are all consistent with two methionines as the heme ligands of this novel type of NEAT heme-binding domain.

Red blood cell hemolysis in sickle cell disease (SCD) releases free hemoglobin. Extracellular hemoglobin and its degradation products, free heme and iron, are highly toxic due to oxidative stress induction and decrease in nitric oxide availability. We propose an approach that helps to eliminate extracellular hemoglobin toxicity in SCD by employing a bacterial protein system that evolved to extract heme from extracellular hemoglobin. NEAr heme Transporter (NEAT) domains from iron-regulated surface determinant proteins from Staphylococcus aureus specifically bind free heme as well as facilitate its extraction from hemoglobin. We demonstrate that a purified NEAT domain fused with human haptoglobin beta-chain is able to remove heme from hemoglobin and reduce heme content and peroxidase activity of hemoglobin. We further use molecular dynamics (MD) simulations to resolve molecular pathway of heme transfer from hemoglobin to NEAT, and to elucidate molecular mechanism of such heme transferring process. Our study is the first of its kind, in which simulations are employed to characterize the process of heme leaving hemoglobin and subsequent rebinding with a NEAT domain. Our MD results highlight important amino acid residues that facilitate heme transfer and will guide further studies for the selection of best NEAT candidate to attenuate free hemoglobin toxicity.

Heme-based gas sensing proteins belong to a group of proteins that are present in signalling pathways of bacteria. A precise regulation of physiological functions, such as intercellular communication or biofilm production, is essential for the survival of these bacteria and their adaptation to the changing surrounding conditions. Heme-based gas sensors are able to detect the concentration of gas molecules in the local environment via their sensory domain (which contains a heme molecule as the intrinsic detection site) and transmit the signal to the functional domain helping to regulate the adaptation of many processes. These, often pathogenic, processes contribute to extended resistance of bacteria against antibiotics. Heme-based sensors are thus potentially a new therapeutic object of interest in antimicrobial treatment. In order to provide this type of treatment, it is crucial to understand the exact mechanism of intramolecular signal transduction facilitated by heme-based sensors. One of the approaches to unravel these mechanisms is further study of model sensory proteins. This thesis focuses on the analysis of a signal transduction performed by two model globin-coupled heme-based oxygen sensors.

O desenvolvimento de antimaláricos é necessario pois, há linhagens de Plasmodium resistentes às drogas em uso e um alvo importante é a via de isoprenoides. Importantes alvos derivados desta via são: heme O já que, há antimaláricos relacionados ao heme; e a giberilina pois, há inibidores desta que não são prejudiciais ao homem. Inabenfide (INA) e uniconazol-P (UNP) inibem a biossíntese de giberilina em plantas e o crescimento de P. falciparum. Inicialmente, identificamos no parasita genes homólogos para a síntese de heme O e A, cox10 e 15, que codificam as enzimas HOS e HAS. Parasitas transgênicos com HOS e HAS marcados com GFP permitiram identificar a localização de cox10 no núcleo e de cox15 na mitocôndria. Identificamos heme O por marcações metabólicas e espectrometria de massa. Entretanto, não identificamos heme A e giberelina. INA e UNP diminuem a biossíntese de heme O e a parasitemia, observado por oxido-redução e marcação metabólica, o que torna a sintese de heme O um interessante alvo para antimaláricos. / The development of antimalarials is necessary because there are Plasmodium strains resistant to the drugs in use and an important target is the isoprenoid pathway. Targets derived from the isoprenoid pathway are: heme O as there are antimalarial drugs related to heme; And gibberillin, because there are inhibitors which are not harmful to man. Inabenfide (INA) and uniconazole-P (UNP) inhibit biosynthesis of gibberillin in plants and of growth the P. falciparum. Initially, we identified in P. falciparum genes homologous to cox10 and 15 encoding the enzymes (HOS and HAS) for synthesis of heme O and A. We created transgenic parasite lines which had HOS and HAS tagged to GFP. These revealed that the subcellular location of cox10 is in the nucleus and of cox15 in mitochondria. We identified heme O by metabolic labeling and mass spectrometry. However, no heme A or gibberellin was detected. INA and UNP decreased heme O biosynthesis and parasitemia as observed by oxido-reduction and metabolic labeling. Our data point to heme O as an important target for antimalarials.

Liu, Jian. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 127-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.