Stefan Hofbauer

Independent evolution of four heme peroxidase superfamilies

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A pattern-based method for establishing a cloud-specific information security management system

Assembling an information security management system (ISMS) according to the ISO 27001 standard is difficult, because the standard provides only very sparse support for system development and documentation. Assembling an ISMS consists of several difficult tasks, e.g., asset identification, threat and risk analysis and security reasoning. Moreover, the standard demands consideration of laws and regulations, as well as privacy concerns. These demands present multi-disciplinary challenges for security engineers. Cloud computing provides scalable IT resources and the challenges of establishing an ISMS increases, because of the significant number of stakeholders and technologies involved and the distribution of clouds among many countries. We analyzed the ISO 27001 demands for these multi-disciplinary challenges and cloud computing systems. Based on these insights, we provide a method that relies upon existing requirements engineering methods and patterns for several security tasks, e.g., context descriptions, threat analysis and policy definition. These can ease the effort of establishing an ISMS and can produce the necessary documentation for an ISO 27001 compliant ISMS. We illustrate our approach using the example of an online bank.

Transiently produced hypochlorite is responsible for the irreversible inhibition of chlorite dismutase.

Chlorite dismutases (Clds) are heme b-containing prokaryotic oxidoreductases that catalyze the reduction of chlorite to chloride with the concomitant release of molecular oxygen. Over time, they are irreversibly inactivated. To elucidate the mechanism of inactivation and investigate the role of the postulated intermediate hypochlorite, the pentameric chlorite dismutase of “Candidatus Nitrospira defluvii” (NdCld) and two variants (having the conserved distal arginine 173 exchanged with alanine and lysine) were recombinantly produced in Escherichia coli. Exchange of the distal arginine boosts the extent of irreversible inactivation. In the presence of the hypochlorite traps methionine, monochlorodimedone, and 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]benzoic acid, the extent of chlorite degradation and release of molecular oxygen is significantly increased, whereas heme bleaching and oxidative modifications of the protein are suppressed. Among other modifications, hypochlorite-mediated formation of chlorinated tyrosines is demonstrated by mass spectrometry. The data obtained were analyzed with respect to the proposed reaction mechanism for chlorite degradation and its dependence on pH. We discuss the role of distal Arg173 by keeping hypochlorite in the reaction sphere for O–O bond formation.

Inactivation of human myeloperoxidase by hydrogen peroxide

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Chlorite dismutases – a heme enzyme family for use in bioremediation and generation of molecular oxygen

Chlorite is a serious environmental concern, as rising concentrations of this harmful anthropogenic compound have been detected in groundwater, drinking water, and soil. Chlorite dismutases (Clds) are therefore important molecules in bioremediation as Clds catalyze the degradation of chlorite to chloride and molecular oxygen. Clds are heme b‐containing oxidoreductases present in numerous bacterial and archaeal phyla. This review presents the phylogeny of functional Clds and Cld‐like proteins, and demonstrates the close relationship of this novel enzyme family to the recently discovered dye‐decolorizing peroxidases. The available X‐ray structures, biophysical and enzymatic properties, as well as a proposed reaction mechanism, are presented and critically discussed. Open questions about structure‐function relationships are addressed, including the nature of the catalytically relevant redox and reaction intermediates and the mechanism of inactivation of Clds during turnover. Based on analysis of currently available data, chlorite dismutase from “Candidatus Nitrospira defluvii” is suggested as a model Cld for future application in biotechnology and bioremediation. Additionally, Clds can be used in various applications as local generators of molecular oxygen, a reactivity already exploited by microbes that must perform aerobic metabolic pathways in the absence of molecular oxygen. For biotechnologists in the field of chemical engineering and bioremediation, this review provides the biochemical and biophysical background of the Cld enzyme family as well as critically assesses Clds technological potential.

Impact of subunit and oligomeric structure on the thermal and conformational stability of chlorite dismutases

Chlorite dismutases (Cld) are unique heme b containing oxidoreductases that convert chlorite to chloride and dioxygen. Recent phylogenetic and structural analyses demonstrated that these metalloproteins significantly differ in oligomeric and subunit structure. Here we have analyzed two representatives of two phylogenetically separated lineages, namely pentameric Cld from Candidatus “Nitrospira defluvii” and dimeric Cld from Nitrobacter winogradskyi having a similar enzymatic activity at room temperature. By application of a broad set of techniques including differential scanning calorimetry, electronic circular dichroism, UV–vis and fluorescence spectroscopy the temperature-mediated and chemical unfolding of both recombinant proteins were analyzed. Significant differences in thermal and conformational stability are reported. The pentameric enzyme is very stable between pH 3 and 10 (Tm = 92 °C at pH 7.0) and active at high temperatures thus being an interesting candidate for bioremediation of chlorite. By contrast the dimeric protein starts to unfold already at 53 °C. The observed unfolding pathways are discussed with respect to the known subunit structure and subunit interaction.

Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes

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Manipulating Conserved Heme Cavity Residues of Chlorite Dismutase: Effect on Structure, Redox Chemistry, and Reactivity

Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from “Candidatus Nitrospira defluvii” (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV–vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O=O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°′ = −113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation.

A stable bacterial peroxidase with novel halogenating activity and an autocatalytically linked heme prosthetic group

Background: First analysis was made of bacterial ancestor of peroxidases from mammalian innate immune system. Results: Highly stable heme enzyme possesses high bromination activity and covalently bound prosthetic group. Conclusion: Post-translational autocatalytic (peroxide-driven) heme modification is found in prokaryotic and eukaryotic halogenating peroxidases. Significance: Peroxidase-mediated production of antimicrobial hypohalous acids was developed early in evolution. Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (−145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.

Redox thermodynamics of high-spin and low-spin forms of chlorite dismutases with diverse subunit and oligomeric structures.

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus “Nitrospira defluvii” (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The E°′ values for free and cyanide-bound proteins were determined to be −119 and −397 mV for NwCld and −113 and −404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism.