Redox thermodynamics of B-class dye-decolorizing peroxidases

With>5000 annotated genes dye-decolorizing peroxidases (DyPs) represent a heme b peroxidase family of broad functional diversity. Bacterial B-class DyPs are poor peroxidases of unknown physiological function. Hydrogen peroxide efficiently mediates the rapid formation of Compound I in B-class DyPs, which, however, is stable and shows modest reactivity towards organic and inorganic electron donors. To understand these characteristics, we have investigated the redox thermodynamics of the one-electron reduction of the ferric high-spin form of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) and the variants D143A, R232A and D143A/R232A. These distal amino acids are fully conserved in all DyPs and play important roles in Compound I formation and maintenance of the heme cavity architecture and substrate access route(s). The E°′ values of the respective redox couples Fe(III)/Fe(II) varied from −350 mV (wild-type KpDyP) to −299 mV (D143A/R232A) at pH 7.0. Variable-temperature spectroelectrochemical experiments revealed that the reduction reaction of B-class DyPs is enthalpically unfavored but entropically favored with significant differences in enthalpic and entropic contributions to E°′ between the four proteins. Molecular dynamics simulations demonstrated the impact of solvent reorganization on the entropy change during reduction reaction and revealed the dynamics and restriction of substrate access channels. Obtained data are discussed with respect to the poor peroxidase activities of B-class DyPs and compared with heme peroxidases from other (super)families as well as with chlorite dismutases, which do not react with hydrogen peroxide but share a similar fold and heme cavity architecture

Compound I formation and reactivity in dimeric chlorite dismutase – Impact of pH and the dynamics of the catalytic arginine

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage

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 wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 \uc5) and the variants D143A (1.3 \uc5), R232A (1.9 \uc5), and D143A/R232A (1.1 \uc5). 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 (LS) cyanide complex as well as on (iv) the stability and reactivity of an oxoiron(IV)porphyrin \u3c0-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

In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase— The Interplay of Key Amino Acids, pH, Buffer and Glycerol - Open Data

Data of In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase— The Interplay of Key Amino Acids, pH, Buffer and Glycerol Authors: Kevin Nys, Vera Pfanzagl, Jeroen Roefs, Christian Obinger and Sabine Van Doorslaer Manuscript ID: ijms-1302052

In vitro heme coordination of a dye-decolorizing peroxidase : the interplay of key amino acids, pH, buffer and glycerol

Dye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function is linked to the heme-pocket architecture, provided the experimental conditions are carefully chosen. Here, these techniques are used to investigate the effect of active site perturbations on the structure of ferric P-class DyP from Klebsiella pneumoniae (KpDyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A). Arg-232 is found to be important for maintaining the heme distal architecture and essential to facilitate an alkaline transition. The latter is promoted in absence of Asp-143. Furthermore, the non-innocent effect of the buffer choice and addition of the cryoprotectant glycerol is shown. However, while unavoidable or indiscriminate experimental conditions are pitfalls, careful comparison of the effects of different exogenous molecules on the electronic structure and spin state of the heme iron contains information about the inherent flexibility of the heme pocket. The interplay between structural flexibility, key amino acids, pH, temperature, buffer and glycerol during in vitro spectroscopic studies is discussed with respect to the poor peroxidase activity of bacterial P-class DyPs

Roles of the distal Asp-Arg pair in Compound I formation of K. pneumoniae dye-decolorizing peroxidase

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6A˚ ) and the variants D143A (1.3 A˚ ), R232A (1.9 A˚ ), and D143A/R232A (1.1 A˚ ). 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-cation radical (Compound I)

The rough endoplasmatic reticulum is the central site of siRNA-mediated RNA silencing

Despite the rapid advancement of our mechanistic understanding of the RNA interference (RNAi) pathways in the past years, the subcellular sites of RNA silencing still remain under debate. Here we show that a lion’s share of transfected small interfering RNA (siRNA) is cleared quickly and only few siRNA molecules are finally getting loaded into Ago2, with as little as 20 30 siRISC molecules per cell sufficient to promote 50 % mRNA knockdown. While the major RNAi pathway proteins are found in most subcellular compartments, the microRNA (miRNA)- and siRNA loaded Ago2 population as well as the RNAi mediated mRNA cleavage product co-sediment exclusively with the membranes of the rough endoplasmatic reticulum (rER) together with the RISC loading complex (RLC) factors Dicer, TRBP and PACT. Moreover, siRNA-loaded Ago2 associates with the cytosolic side of membranes through TRBP and PACT in an RNA-independent manner, potentially mediated through indirect interaction via Dicer. Our findings demonstrate that the outer membrane of the rER is the central site of RNA silencing, which explains the remarkable thermodynamic and kinetic efficiency of this mechanism

Arresting the Catalytic Arginine in Chlorite Dismutases: Impact on Heme Coordination, Thermal Stability, and Catalysis

Articles ASAP (as soon as publishable) posted online and available to view immediately after technical editing, formatting for publication, and author proofing.Abstract Chlorite dismutases (Clds) are heme b-containing oxidoreductases that can decompose chlorite to chloride and molecular oxygen. They are divided in two clades that differ in oligomerization, subunit architecture, and the hydrogen-bonding network of the distal catalytic arginine, which is proposed to switch between two conformations during turnover. To understand the impact of the conformational dynamics of this basic amino acid on heme coordination, structure, and catalysis, Cld from Cyanothece sp. PCC7425 was used as a model enzyme. As typical for a clade 2 Cld, its distal arginine 127 is hydrogen-bonded to glutamine 74. The latter has been exchanged with either glutamate (Q74E) to arrest R127 in a salt bridge or valine (Q74V) that mirrors the setting in clade 1 Clds. We present the X-ray crystal structures of Q74V and Q74E and demonstrate the pH-induced changes in the environment and coordination of the heme iron by ultraviolet–visible, circular dichroism, and electron paramagnetic resonance spectroscopies as well as differential scanning calorimetry. The conformational dynamics of R127 is shown to have a significant role in heme coordination during the alkaline transition and in the thermal stability of the heme cavity, whereas its impact on the catalytic efficiency of chlorite degradation is relatively small. The findings are discussed with respect to (i) the flexible loop connecting the N-terminal and C-terminal ferredoxin-like domains, which differs in clade 1 and clade 2 Clds and carries Q74 in clade 2 proteins, and (ii) the proposed role(s) of the arginine in catalysis

Arresting the catalytic arginine in chlorite dismutases : impact on heme coordination, thermal stability, and catalysis

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that can decompose chlorite to chloride and molecular oxygen. They are divided in two clades that differ in oligomerization, subunit architecture, and the hydrogen-bonding network of the distal catalytic arginine, which is proposed to switch between two conformations during turnover. To understand the impact of the conformational dynamics of this basic amino acid on heme coordination, structure, and catalysis, Cld from Cyanothece sp. PCC7425 was used as a model enzyme. As typical for a clade 2 Cld, its distal arginine 127 is hydrogen-bonded to glutamine 74. The latter has been exchanged with either glutamate (Q74E) to arrest R127 in a salt bridge or valine (Q74V) that mirrors the setting in clade 1 Clds. We present the X-ray crystal structures of Q74V and Q74E and demonstrate the pH-induced changes in the environment and coordination of the heme iron by ultraviolet–visible, circular dichroism, and electron paramagnetic resonance spectroscopies as well as differential scanning calorimetry. The conformational dynamics of R127 is shown to have a significant role in heme coordination during the alkaline transition and in the thermal stability of the heme cavity, whereas its impact on the catalytic efficiency of chlorite degradation is relatively small. The findings are discussed with respect to (i) the flexible loop connecting the N-terminal and C-terminal ferredoxin-like domains, which differs in clade 1 and clade 2 Clds and carries Q74 in clade 2 proteins, and (ii) the proposed role(s) of the arginine in catalysis