Unspecific peroxygenases (UPOs) are highly versatile biocatalysts capable of removing various persistent environmental contaminants and performing sustainable chemical transformations. These oxidoreductases contain heme b as their prosthetic group. As all classical peroxidases, they are activated by the molecules of hydrogen peroxide to incorporate the oxygen atom into numerous organic molecules. Alternatively, they can use ascorbate as a cosubstrate. In sequence databases an ever-increasing number of their DNA and protein sequences occurs. Reconstructed molecular phylogeny of the corresponding peroxidase-peroxygenase superfamily reveals a high diversity of gene distribution for UPOs in the whole kingdom of fungi. A majority of identified UPO sequences stems from numerous species of Dikarya. Although members of this superfamily were recently detected also in early diverging fungal lineages, UPOs from the phyla of Mucoromycota, Glomeromycota and Chytridiomycota remain not sufficiently investigated. Moreover, newly discovered genes coding for UPOs were recently identified also among early diverging eukaryotic lineages of amoebas and green algae in various biotops. With a large palette of potential substrates these oxidoreductases serve as a versatile tool in enzyme catalysed synthetic reactions, but their real physiological substrates need to be recognized in the future. Most important among self-sufficient UPO-catalysed reactions are oxyfunctionalizations of various aliphatic and aromatic molecules. In this critical review an outlook is given for investigation and engineering of novel UPO variants including products of directed evolution. Future research on UPOs shall be mainly focused on basal fungal and emerging non-fungal sources for their promising applications in environmentally friendly technologies.

All phytopathogenic fungi have two catalase-peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase-peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV-Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a K d value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure-function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host-pathogen interaction.