Graphical abstract

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Although structure and steady-state kinetics of Clds have been elucidated, many questions remain (e.g., the mechanism of chlorite cleavage and the pH dependence of the reaction). Here, we present high-resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple, and the UV–vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite do not recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during turnover. Homolytic cleavage of chlorite is proposed.

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.

Heme biosynthesis in Gram‐positive bacteria follows a recently described coproporphyrin‐dependent pathway with HemQ catalyzing the decarboxylation of coproheme to heme b. Here we present the first crystal structure of a HemQ (homopentameric coproheme‐HemQ from Listeria monocytogenes) at 1.69 Å resolution and the conversion of coproheme to heme b followed by UV‐vis and resonance Raman spectroscopy as well as mass spectrometry. The ferric five‐coordinated coproheme iron of HemQ is weakly bound by a neutral proximal histidine H174. In the crystal structure of the resting state, the distal Q187 (conserved in Firmicutes HemQ) is H‐bonded with propionate p2 and the hydrophobic distal cavity lacks solvent water molecules. Two H2O2 molecules are shown to be necessary for decarboxylation of the propionates p2 and p4, thereby forming the corresponding vinyl groups of heme b. The overall reaction is relatively slow (k cat/KM = 1.8 × 102 m −1·s−1 at pH 7.0) and occurs in a stepwise manner with a three‐propionate intermediate. We present the noncovalent interactions between coproheme and the protein and propose a two‐step reaction mechanism. Furthermore, the structure of coproheme‐HemQ is compared to that of the phylogenetically related heme b‐containing chlorite dismutases.DatabaseStructural data are available in the PDB under the accession number 5LOQ.

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

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 Cld's technological potential.

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.

Graphical abstract