Heme is an iron-containing modified tetrapyrrole ubiquitous in nature. It is the most versatile metalloprosthetic group being involved in diverse essential functions including electron transport, catalysis, gas sensing and transport, signalling  and transcription. The heme cofactor makes it possible to carry out manifold and specific biochemical redox reactions in an efficient and safe manner. Thus heme enzymes have been the object of research for many decades.

In our research we aim at understanding structure-function relationships and the molecular reaction mechanism of heme oxidoreductases of prokaryotic and eukaryotic origin. In detail we aim at understanding (i) the kinetics and thermodynamics of electron transfer reactions, (ii) the impact of the heme cavity architecture and posttranslational modification of heme and protein on catalysis, and, finally, (iii) the physiological role of those biocatalysts. We use a broad range of methods including recombinant production of wild-type and mutant proteins in E. coli, Pichia pastoris, HEK or CHO cell lines, UV-VIS-, circular dichroism- and resonance Raman spectroscopies, static and dynamic light scattering, multi-mixing stopped-flow spectroscopy, spectroelectrochemistry, differential scanning and isothermal titration calorimetry, mass spectrometry as well as X-ray and neutron crystallography.


Currently we are working on following topics:

Biochemistry of human peroxidases (myeloperoxidase, lactoperoxidase, eosinophil peroxidase)

Structure-function relationships of human peroxidases are investigated in order to understand their role in immunology and inflammation. Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are isolated from leukocytes (neutrophils and eosinophils, respectively), whereas lactoperoxidase (LPO) is isolated from bovine milk. Recombinant wild-type and mutant proteins are heterologously overexpressed in CHO- and HEK cell lines. The main objective is to understand the mechanism of one- and two-electron oxidation of inorganic and organic substrates and the impact of posttranslational heme modification (i.e. formation of covalent heme to protein ester and sulfonium ion bonds) on those redox reactions. Furthermore, we design and test specific mechanism-based inhibitors of MPO and EPO and investigate the interaction of human heme peroxidases with hydrogen sulphide that is gaining attention as an important element in sulphide-mediated protection against oxidative stress as well as in regulation of redox signalling.

Furthermore, current work focuses on the biochemistry of homologous peroxidases in lower eukaryotes (e.g. Dictyostelium discoideum) or bacteria.

Biochemistry of Peroxidasins

Together with MPO, EPO or LPO, peroxidasins belong to the peroxidase-cyclooxygenase superfamily (i.e. heme peroxidases with posttranslational modified heme). Peroxidasins are homotrimeric multidomain peroxidases, which are secreted to the extracellular matrix (ECM) and release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds for reinforcement of collagen IV in basement membranes. We aim at understanding the role of the various domains in the interaction with ECM proteins and at solving the high resolution structure of human peroxidasin 1. Furthermore, we try to understand the molecular mechanism of one- and two-electron oxidation reactions and the role of the multidomain architecture on the (controlled) release of mobile oxidants like HOBr.

Moreover, functional and structural studies on invertebrate and vertebrate peroxidasins will give new insights into the evolution of this protein family as well as its proposed roles in innate immunity and/or extracellular matrix stabilization.

Structural and mechanistic studies on a dimeric chlorite dismutase

Some microorganisms can deal with perchlorate and chlorate. The key enzyme in these (per)chlorate-reducing bacteria is the heme enzyme chlorite dismutase (Cld) that finally detoxifies harmful chlorite by its conversion into chloride and dioxygen.

Based on our successful elucidation of the X-ray (and neutron) structures of dimeric Clds from Cyanothece sp. PCC7425 and Nitrobacter winogradskyi and pentameric Cld from Nitrospira defluvii, we aim at understanding (i) the molecular mechanism of enzyme oxidation by chlorite, (ii) the electronic structures and spectral signatures of the involved redox intermediates, (iii) the (unique) mechanism of O-O bond formation and (iv) the molecular basis of observed differences in the kinetics of chlorite degradation between dimeric and pentameric Clds. Obtained data will provide the basis for application of Clds in bioremediation of harmful anthropogenic chlorite found in groundwater, drinking water, and soils.

Biochemistry of coproheme decarboxylase and the coproporphyrin-dependent heme biosynthesis pathway

In 2015 the so-called “coproporphyrin-dependent” heme biosynthesis pathway was described for the first time. It is mainly utilized by Gram-positive (monoderm) bacteria and differs from the classical (protoporphyrin-dependent) pathway found in diderm bacteria and eukaryotes. Our research focuses on the molecular mechanism of the radicalic, hydrogen-peroxide dependent two-step decarboxylation of coproheme (iron coproporphyrin III) thereby releasing two CO2 molecules and heme b. We focus on coproheme decarboxylase (ChdC) from pathogenic bacteria including Listeria monocytogenes, Staphylococcus aureus, and Corynebacterium diphteriae.

Further we are interested in the bigger picture and expand our research to the entire coproporphyrin-dependent heme biosynthesis pathway and investigate in addition the enzymology of uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and coproheme ferrochelatase.

Structure-function relationship and physiological role of so-called “dye-decolourizing peroxidases”

Dye-decolourizing peroxidases (DyPs) were originally described to be able to decolourize bulky dyes and oxidize various one-electron donors. Phylogenetically this family of heme enzymes can be classified into three main groups. Our research focuses on A- and B-type DyPs, which exhibit a poor oxidation capacity of dyes or of classical artificial organic and inorganic peroxidase substrates. In detail we study DyPs from Klebsiella pneumoniae and Escherichia coli. Especially, B-type DyPs are mostly unreactive towards known electron donors and form a highly stable Compound I. The physiological role is under discussion.

We are interested in the mechanistic details of Compound I formation as well as in its high resolution structure and search for potential interaction partners including a redox-active ion channel.

Collaboration partners (alphabetical order)

Gianantonio Battistuzzi
Universitá di Modena e Reggio Emilia, Dipartimento Scienze Chimiche e Geologiche, Italy

Michael Davies
University of Copenhagen, Department of Biomedical Sciences, Denmark

Kristina Djinović-Carugo
Universität Wien, Max. F. Perutz Laboratories, Department of Structural and Computational Biology, Austria

Tony Kettle
University of Otago, Centre for Free Radical Research, New Zealand

Peter Nagy
National Institute of Oncology, Department of Molecular Immunology and Toxicology, Hungary

Chris Oostenbrink
University of Natural Resources and Life Sciences Vienna, Department of Material Sciences and Process Engineering, Austria

Giulietta Smulevich
Universitá di Firenze, Dipartimento di Chimica “Ugo Schiff”, Italy

Thierry Soldati
University of Geneva, Department of Biochemistry, Switzerland

Pierre van Antwerpen
Université Libre de Bruxelles, Department of Pharmacy, Belgium

Sabine van Doorslaer
University of Antwerp, Department of Physics, Belgium