Theoretical studies of mononuclear non-heme iron active sites

The quantum chemical investigations presented in this thesis use hybrid density functional theory to shed light on the catalytic mechanisms of mononuclear non-heme iron oxygenases, accommodating a ferrous ion in their active sites. More specifically, the dioxygen activation process and the subsequent oxidative reactions in the following enzymes were studied: tetrahydrobiopterin-dependent hydroxylases, naphthalene 1,2-dioxygenase and α-ketoglutarate-dependent enzymes. In light of many experimental efforts devoted to the functional mimics of non-heme iron oxygenases, the reactivity of functional analogues was also examined. The computed energetics and the available experimental data served to assess the feasibility of the reaction mechanisms investigated. Dioxygen activation in tetrahydrobiopterin- and α-ketoglutarate-dependent enzymes were found to involve a high-valent iron-oxo species, which was then capable of substrate hydroxylation. In the case of naphthalene 1,2-dioxygenase, the reactivity of an iron(III)-hydroxperoxo species toward the substrate was investigated and compared to the biomimetic counterpart.

Vibrational spectroscopic and electrochemical investigations of multi-centered heme proteins in biomimetic membrane architectures

Membrane proteins play a major role in every living cell. They are the key factors in the cell’s metabolism and in other functions, for example in cell-cell interaction, signal transduction, and transport of ions and nutrients. Cytochrome c oxidase (CcO), as one of the membrane proteins of the respiratory chain, plays a significant role in the energy transformation of higher organisms. CcO is a multi centered heme protein, utilizing redox energy to actively transport protons across the mitochondrial membrane. One aim of this dissertation is to investigate single steps in the mechanism of the ion transfer process coupled to electron transfer, which are not fully understood. The protein-tethered bilayer lipid membrane is a general approach to immobilize membrane proteins in an oriented fashion on a planar electrode embedded in a biomimetic membrane. This system enables the combination of electrochemical techniques with surface enhanced resonance Raman (SERRS), surface enhanced reflection absorption infrared (SEIRAS), and surface plasmon spectroscopy to study protein mediated electron and ion transport processes. The orientation of the enzymes within the surface confined architecture can be controlled by specific site-mutations, i.e. the insertion of a poly-histidine tag to different subunits of the enzyme. CcO can, thus, be oriented uniformly with its natural electron pathway entry pointing either towards or away from the electrode surface. The first orientation allows an ultra-fast direct electron transfer(ET) into the protein, not provided by conventional systems, which can be leveraged to study intrinsic charge transfer processes. The second orientation permits to study the interaction with its natural electron donor cytochrome c. Electrochemical and SERR measurements show conclusively that the redox site structure and the activity of the surface confined enzyme are preserved. Therefore, this biomimetic system offers a unique platform to study the kinetics of the ET processes in order to clarify mechanistic properties of the enzyme. Highly sensitive and ultra fast electrochemical techniques allow the separation of ET steps between all four redox centres including the determination of ET rates. Furthermore, proton transfer coupled to ET could be directly measured and discriminated from other ion transfer processes, revealing novel mechanistic information of the proton transfer mechanism of cytochrome c oxidase. In order to study the kinetics of the ET inside the protein, including the catalytic center, time resolved SEIRAS and SERRS measurements were performed to gain more insight into the structural and coordination changes of the heme environment. The electrical behaviour of tethered membrane systems and membrane intrinsic proteins as well as related charge transfer processes were simulated by solving the respective sets of differential equations, utilizing a software package called SPICE. This helps to understand charge transfer processes across membranes and to develop models that can help to elucidate mechanisms of complex enzymatic processes.

Time-resolved surface enhanced resonance Raman spectro-electrochemistry of heme proteins

The membrane protein Cytochrome c Oxidase (CcO) is one of the most important functional bio-molecules. It appears in almost every eukaryotic cell and many bacteria. Although the different species differ in the number of subunits, the functional differences are merely marginal. CcO is the terminal link in the electron transfer pathway of the mitochondrial respiratory chain. Electrons transferred to the catalytic center of the enzyme conduce to the reduction of molecular oxygen to water. Oxygen reduction is coupled to the pumping of protons into the inter-membrane space and hence generates a difference in electrochemical potential of protons across the inner mitochondrial membrane. This potential difference drives the synthesis of adenosine triphosphate (ATP), which is the universal energy carrier within all biological cells. rnrnThe goal of the present work is to contribute to a better understanding of the functional mechanism of CcO by using time-resolved surface enhanced resonance Raman spectroscopy (TR-SERRS). Despite intensive research effort within the last decades, the functional mechanism of CcO is still subject to controversial discussions. It was the primary goal of this dissertation to initiate electron transfer to the redox centers CuA, heme a, heme a3 and CuB electrochemically and to observe the corresponding redox transitions in-situ with a focus on the two heme structures by using SERRS. A measuring cell was developed, which allowed combination of electrochemical excitation with Raman spectroscopy for the purpose of performing the accordant measurements. Cytochrome c was used as a benchmark system to test the new measuring cell and to prove the feasibility of appropriate Raman measurements. In contrast to CcO the heme protein cc contains only a single heme structure. Nevertheless, characteristic Raman bands of the hemes can be observed for both proteins.rnrnIn order to investigate CcO it was immobilized on top of a silver substrate and embedded into an artificial membrane. The catalytic activity of CcO and therefore the complete functional capability of the enzyme within the biomimetic membrane architecture was verified using cyclic voltammetry. Raman spectroscopy was performed using a special nano-structured silver surface, which was developed within the scope of the present work. This new substrate combined two fundamental properties. It facilitated the formation of a protein tethered bilayer lipid membrane (ptBLM) and it allowed obtaining Raman spectra with sufficient high signal-to-noise ratios.rnSpectro-electrochemical investigations showed that at open circuit potential the enzyme exists in a mixed-valence state, with heme a and and heme a3 in the reduced and oxidized state, respectively. This was considered as an intermediate state between the non-activated and the fully activated state of CcO. Time-resolved SERRS measurements revealed that a hampered electron transfer to the redox center heme a3 characterizes this intermediate state.rn

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.

Heme oxygenase-1 is a critical regulator of nitric oxide production in enterohemorrhagic Escherichia coli-infected human enterocytes

Enterohemorrhagic Escherichia coli (EHEC) are the causative agent of hemolytic-uremic syndrome. In the first stage of the infection, EHEC interact with human enterocytes to modulate the innate immune response. Inducible NO synthase (iNOS)-derived NO is a critical mediator of the inflammatory response of the infected intestinal mucosa. We therefore aimed to analyze the role of EHEC on iNOS induction in human epithelial cell lines. In this regard, we show that EHEC down-regulate IFN-gamma-induced iNOS mRNA expression and NO production in Hct-8, Caco-2, and T84 cells. This inhibitory effect occurs through the decrease of STAT-1 activation. In parallel, we demonstrate that EHEC stimulate the rapid inducible expression of the gene hmox-1 that encodes for the enzyme heme oxygenase-1 (HO-1). Knock-down of hmox-1 gene expression by small interfering RNA or the blockade of HO-1 activity by zinc protoporphyrin IX abrogated the EHEC-dependent inhibition of STAT-1 activation and iNOS mRNA expression in activated human enterocytes. These results highlight a new strategy elaborated by EHEC to control the host innate immune response.

Ischemia-induced up-regulation of heme oxygenase-1 protects from apoptotic cell death and tissue necrosis

BACKGROUND: Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. MATERIALS AND METHODS: In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. RESULTS: Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). CONCLUSION: Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite

Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well as Plasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains.

Mesenchymal stromal cells improve survival during sepsis in the absence of heme oxygenase-1: the importance of neutrophils

The use of mesenchymal stromal cells (MSCs) for treatment of bacterial infections, including systemic processes like sepsis, is an evolving field of investigation. This study was designed to investigate the potential use of MSCs, harvested from compact bone, and their interactions with the innate immune system, during polymicrobial sepsis induced by cecal ligation and puncture (CLP). We also wanted to elucidate the role of endogenous heme oxygenase (HO)-1 in MSCs during a systemic bacterial infection. MSCs harvested from the bones of HO-1 deficient (-/-) and wild-type (+/+) mice improved the survival of HO-1(-/-) and HO-1(+/+) recipient mice when administered after the onset of polymicrobial sepsis induced by CLP, compared with the administration of fibroblast control cells. The MSCs, originating from compact bone in mice, enhanced the ability of neutrophils to phagocytize bacteria in vitro and in vivo and to promote bacterial clearance in the peritoneum and blood after CLP. Moreover, after depleting neutrophils in recipient mice, the beneficial effects of MSCs were entirely lost, demonstrating the importance of neutrophils for this MSC response. MSCs also decreased multiple organ injury in susceptible HO-1(-/-) mice, when administered after the onset of sepsis. Taken together, these data demonstrate that the beneficial effects of treatment with MSCs after the onset of polymicrobial sepsis is not dependent on endogenous HO-1 expression, and that neutrophils are crucial for this therapeutic response.

Ketamine-induced hepatoprotection: the role of heme oxygenase-1.

Lipopolysaccharide (LPS) causes hepatic injury that is mediated, in part, by upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Ketamine has been shown to prevent these effects. Because upregulation of heme oxygenase-1 (HO-1) has hepatoprotective effects, as does carbon monoxide (CO), an end product of the HO-1 catalytic reaction, we examined the effects of HO-1 inhibition on ketamine-induced hepatoprotection and assessed whether CO could attenuate LPS-induced hepatic injury. One group of rats received ketamine (70 mg/kg ip) or saline concurrently with either the HO-1 inhibitor tin protoporphyrin IX (50 micromol/kg ip) or saline. Another group of rats received inhalational CO (250 ppm over 1 h) or room air. All rats were given LPS (20 mg/kg ip) or saline 1 h later and euthanized 5 h after LPS or saline. Liver was collected for iNOS, COX-2, and HO-1 (Western blot), NF-kappaB and PPAR-gamma analysis (EMSA), and iNOS and COX-2 mRNA analysis (RT-PCR). Serum was collected to measure alanine aminotransferase as an index of hepatocellular injury. HO-1 inhibition attenuated ketamine-induced hepatoprotection and downregulation of iNOS and COX-2 protein. CO prevented LPS-induced hepatic injury and upregulation of iNOS and COX-2 proteins. Although CO abolished the ability of LPS to diminish PPAR-gamma activity, it enhanced NF-kappaB activity. These data suggest that the hepatoprotective effects of ketamine are mediated primarily by HO-1 and its end product CO.

Crystal structure of a multifunctional 2-Cys peroxiredoxin heme-binding protein 23 kDa/proliferation-associated gene product

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Replacement of the proximal heme thiolate ligand in chloroperoxidase with a histidine residue

Chloroperoxidase is a versatile heme enzyme which can cross over the catalytic boundaries of other oxidative hemoproteins and perform multiple functions. Chloroperoxidase, in addition to catalyzing classical peroxidative reactions, also acts as a P450 cytochrome and a potent catalase. The multiple functions of chloroperoxidase must be derived from its unique active site structure. Chloroperoxidase possesses a proximal cysteine thiolate heme iron ligand analogous to the P450 cytochromes; however, unlike the P450 enzymes, chloroperoxidase possesses a very polar environment distal to its heme prosthetic group and contains a glutamic acid residue in close proximity to the heme iron. The presence of a thiolate ligand in chloroperoxidase has long been thought to play an essential role in its chlorination and epoxidation activities; however, the research reported in this paper proves that hypothesis to be invalid. To explore the role of Cys-29, the amino acid residue supplying the thiolate ligand in chloroperoxidase, Cys-29 has been replaced with a histidine residue. Mutant clones of the chloroperoxidase genome have been expressed in a Caldariomyces fumago expression system by using gene replacement rather than gene insertion technology. C. fumago produces wild-type chloroperoxidase, thus requiring gene replacement of the wild type by the mutant gene. To the best of our knowledge, this is the first time that gene replacement has been reported for this type of fungus. The recombinant histidine mutants retain most of their chlorination, peroxidation, epoxidation, and catalase activities. These results downplay the importance of a thiolate ligand in chloroperoxidase and suggest that the distal environment of the heme active site plays the major role in maintaining the diverse activities of this enzyme.

A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme

Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14α-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14α-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.