**3. Heme-Iron-Dependent Haloperoxidases**

The heme-iron-dependent haloperoxidases were the first and most intensively studied haloperoxidases. Back in the 1960s, an enzyme from the fungus *Caldariomyces fumago* (*Leptomyxes fumago*) was shown to be responsible for the halogenation of 1,3-cyclopentadion to the natural compound caldariomycin (**9**) [53]. Upon further investigation, it could be shown that it contained a heme-prosthetic group tethered to the enzyme by a distal cysteine ligand, very similar to the P450 monooxygenases [54].

The catalytic cycle (Figure 7) displays a key intermediate, the FeIV-oxo-species, to oxidize chlorine to hypochlorite, which is released and may be attacked by an electron-rich substrate serving as an electrophile. In presence of excess hydrogen peroxide, this complex can alternatively decompose to molecular oxygen and chloride.

**Figure 7.** Proposed catalytic cycle of heme-iron-dependent haloperoxidases, shown on the example of CPO from *C. fumago*. In the resting state (3 o' clock), water is bound to the heme-iron, which is subsequently replaced by hydrogen peroxide. After protonation of this complex by a catalytic glutamate (Glu183), water is eliminated, creating the actual active species, the Fe(IV)-oxo complex. A halide, in this case chloride, binds to the Fe(IV)-oxo species and is released as hypochloric acid, regenerating the heme-site by hydrolysis with water. Alternatively, another molecule hydrogen peroxide may attack, leading to the disproportion of the complex to molecular oxygen, water, and chloride [54,55].

As the enzyme resembled characteristics from peroxidases as well as monooxygenases, it was classified as a heme-iron-dependent haloperoxidase and due to its ability to oxidize all halides besides fluorine was named chloroperoxidase. Recently it was revealed that actually two *Cf*-*cpo* genes within the *C. fumago* genome exist, sharing a high sequence identity and both being present in the secreted supernatant of its host [56]. Since its discovery, the enzyme was target of many mechanistic and biocatalytic studies. To much surprise, the formed hypohalous acid does not leave the active site freely, but is held back by amino acids placed in the halide entrance tunnel of *Cf*-CPO, allowing for regio- and enantioselectivity to a certain degree, mainly depending on the nature of the substrate [57]. Its major drawback, however, was the oxidative inactivation every heme-iron-containing protein suffers after exposure to oxygen as well as a high sensitivity for high hydrogen peroxide concentrations. As the genetic modification of the fungus can prove tedious, the application of this enzyme in biocatalysis might seem limited, however due to the fruitful work of Pickard et al., protocols are available for a reasonable production and secretion of the enzyme in the native host, *C. fumago* [58].

As a catalyst, *Cf*-CPO was shown to be rather robust und allow a variety of different organic transformations, where some are not always bound to a halogenating step. It could be applied in cascade reactions with oxidases leading to halocyclization reactions of allenes (**10**) and even be immobilized for (semi-)continuous-flow bioreactors [59–61] (see Figure 8). It was used for the halogenation of phenolic monoterpenes like thymol (**12**) and carvacrol, excelling with drastically lower catalyst loadings (by five orders of magnitude) compared to chemical alternatives like CuII-catalysis [59]. Furthermore, it was shown to be capable of halogenating *trans*-cinnamic acid and other unsaturated carboxylic acids, as well as catalyze enantioselective epoxidation of alkenes [62,63]. One bottleneck observed was the low substrate loading, impairing possible preparative applicability.

**Figure 8.** Example reactions of *Cf-*CPO involved in biocatalytic conversions of organic molecules (**A**): Cyclization of allenes (**10**) to the product **11** induced by halogenation with Br− by *Cf-*CPO. (**B**): Unselective chlorination of thymol (**12**) by *Cf-*CPO.

Besides chloroperoxidase from *C. fumago*, not many members of this subclass have been dealt with. The bromoperoxidases from *Pseudomonas aureofaciens* and *Penicillus capitatus* are other examples of such heme-iron-dependent enzymes [64,65]. However, beside classic characterization experiments, revealing similar properties to *Cf*-CPO such as high thermal stability and sensitivity to high hydrogen peroxide concentrations, no complex biotransformations were investigated with these enzymes, yet (see Table 1) [66].


**Table 1.** Enzymological properties of heme-dependent haloperoxidases (\* original host).
