Distal Arginine Research Articles

Role of distal arginine residue in the mechanism of heme nitrite reductases.

Heme nitrite reductases reduce NO2- by 1e-/2H+ to NO or by 6e-/8H+ to NH4+ which are key steps in the global nitrogen cycle. Second-sphere residues, such as arginine (with a guanidine head group), are proposed to play a key role in the reaction by assisting substrate binding and hydrogen bonding and by providing protons to the active site for the reaction. The reactivity of an iron porphyrin with a NO2- covalently attached to a guanidinium arm in its 2nd sphere was investigated to understand the role of arginine residues in the 2nd sphere of heme nitrite reductases. The presence of the guanidinium residue allows the synthetic ferrous porphyrin to reduce NO2- and produce a ferrous nitrosyl species ({FeNO}7), where the required protons are provided by the guanidinium group in the 2nd sphere. However, in the presence of additional proton sources in solution, the reaction of ferrous porphyrin with NO2- results in the formation of ferric porphyrin and the release of NO. Spectroscopic and kinetic data indicated that re-protonation of the guanidine group in the 2nd sphere by an external proton source causes NO to dissociate from a ferric nitrosyl species ({FeNO}6) at rates similar to those observed for enzymatic sites. This re-protonation of the guanidine group mimics the proton recharge mechanism in the active site of NiR. DFT calculations indicated that the lability of the Fe-NO bond in the {FeNO}6 species is derived from the greater binding affinity of anions (e.g. NO2-) to the ferric center relative to neutral NO due to hydrogen bonding and electrostatic interaction of these bound anions with the protonated guanidium group in the 2nd sphere. The reduced {FeNO}7 species, once formed, is not affected significantly by the re-protonation of the guanidine residue. These results provide direct insight into the role of the 2nd sphere arginine residue present in the active sites of heme-based NiRs in determining the fate of NO2- reduction. Specifically, the findings using the synthetic model suggest that rapid re-protonation of these arginine residues may trigger the dissociation of NO from the {FeNO}6, which may also be the case in the protein active site.

Switchable Enzyme-mimicking catalysts Self-Assembled from de novo designed peptides and DNA G-quadruplex/hemin complex.

Reconstruction of enzymatic active site in an artificial system is key to achieving high catalytic efficiency. Herein, we report the self-assembly of the lysine-containing peptides with guanine-rich DNA and hemin to form peroxidase-mimicking active sites and catalytic nanoparticles. The DNA strand self-folds into a G-quadruplex structure that provides a supramolecular scaffold and a potential axial ligand for hemin. The β-sheet forming capability of the lysine-containing peptides is found to affect the catalytic synergy between the G-quadruplex DNA and the peptide. It is hypothesized that the β-sheet formation of the peptides results in the enrichment of the lysine residues, which distribute on the distal side of hemin to promote the formation of Compound I, like distal arginine residue in natural heme pocket. Incorporation of the histidine residues into the lysine-containing peptides further enhanced the hemin activities, indicating the cooperation between the lysine and histidine. Furthermore, the peptide/DNA/hemin complexes can be switched between active and inactive state by reversible formation and deformation of the DNA G-quadruplex, which was attributed to the peptides-promoted conformational changes of the DNA components. This work opens an avenue to mimic the catalytic residues and their spatial distribution in the natural enzymes, and shed light on the design of the smart biocatalysts that can respond to the environmental stimuli.

Structural Characterization of Cytochrome c′β‐Met from an Ammonia-Oxidizing Bacterium

The ammonia-oxidizing bacterium Nitrosomonas europaea expresses two cytochromes in the P460 superfamily that are predicted to be structurally similar. In one, cytochrome (cyt) P460, the substrate hydroxylamine (NH2OH) is converted to nitric oxide (NO) and nitrous oxide (N2O) requiring a unique heme-lysyl cross-link in the catalytic cofactor. In the second, cyt , the cross-link is absent, and the cytochrome instead binds H2O2 forming a ferryl species similar to compound II of peroxidases. Here, we report the 1.80 Å crystal structure of cyt ─a well-expressed protein in N. europaea with a lysine to a methionine replacement at the cross-linking position. The structure of cyt is characterized by a large β-sheet typical of P460 members; however, several localized structural differences render cyt distinct. This includes a large lasso-like loop at the "top" of the cytochrome that is not observed in other structurally characterized members. Active site variation is also observed, especially in comparison to its closest homologue cyt from the methane-oxidizing Methylococcus capsulatus Bath, which also lacks the cross-link. The phenylalanine "cap" which is presumed to control small ligand access to the distal heme iron is replaced with an arginine, reminiscent of the strictly conserved distal arginine in peroxidases and to the NH2OH-oxidizing cytochromes P460. A critical proton-transferring glutamate residue required for NH2OH oxidation is nevertheless missing in the active site. This in part explains the inability of cyt to oxidize NH2OH. Our structure also rationalizes the absence of a methionyl cross-link, although the side chain's spatial position in the structure does not eliminate the possibility that it could form under certain conditions.

Dual arginine recognition of LRRK2 phosphorylated Rab GTPases

Parkinson’s-disease-associated LRRK2 is a multidomain Ser/Thr kinase that phosphorylates a subset of Rab GTPases to control their effector functions. Rab GTPases are the prime regulators of membrane trafficking in eukaryotic cells. Rabs exert their biological effects by recruitment of effector proteins to subcellular compartments via their Rab-binding domain (RBD). Effectors are modular and typically contain additional domains that regulate various aspects of vesicle formation, trafficking, fusion, and organelle dynamics. The RBD of effectors is typically an α-helical coiled coil that recognizes the GTP conformation of the switch 1 and switch 2 motifs of Rabs. LRRK2 phosphorylates Rab8a at T72 (pT72) of its switch 2 α-helix. This post-translational modification enables recruitment of RILPL2, an effector that regulates ciliogenesis in model cell lines. A newly identified RBD motif of RILPL2, termed the X-cap, has been shown to recognize the phosphate via direct interactions between an arginine residue (R132) and pT72 of Rab8a. Here, we show that a second distal arginine (R130) is also essential for phospho-Rab binding by RILPL2. Through structural, biophysical, and cellular studies, we find that R130 stabilizes the primary R132:pT72 salt bridge through favorable enthalpic contributions to the binding affinity. These findings may have implications for the mechanism by which LRRK2 activation leads to assembly of phospho-Rab complexes and subsequent control of their membrane trafficking functions in cells.

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.

Serial Femtosecond Zero Dose Crystallography Captures a Water‐Free Distal Heme Site in a Dye‐Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV=O Formation

AbstractObtaining structures of intact redox states of metal centers derived from zero dose X‐ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye‐decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV=O and a porphyrin cation radical). Using serial femtosecond X‐ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV=O redox states of a B‐type DyP. These structures reveal a water‐free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.

Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV =O Formation.

Obtaining structures of intact redox states of metal centers derived from zero dose X‐ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye‐decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV=O and a porphyrin cation radical). Using serial femtosecond X‐ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV=O redox states of a B‐type DyP. These structures reveal a water‐free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.

Biochemical features of dye-decolorizing peroxidases: Current impact on lignin degradation.

Dye-decolorizing peroxidases (DyP) were originally discovered in fungi for their ability to decolorize several different industrial dyes. DyPs catalyze the oxidation of a variety of substrates such as phenolic and nonphenolic aromatic compounds. Catalysis occurs in the active site or on the surface of the enzyme depending on the size of the substrate and on the existence of radical transfer pathways available in the enzyme. DyPs show the typical features of heme-containing enzymes with a Soret peak at 404-408 nm. They bind hydrogen peroxide that leads to the formation of the so-called Compound I, the key intermediate for catalysis. This then decays into Compound II yielding back Fe(III) at its resting state. Each catalytic cycle uses two electrons from suitable electron donors and generates two product molecules. DyPs are classified as a separate class of peroxidases. As all peroxidases they encompass a conserved histidine that acts as the fifth heme ligand, however all primary DyP sequences contain a conserved GxxDG motif and a distal arginine that is their characteristic. Given their ability to attack monomeric and dimeric lignin model compounds as well as polymeric lignocellulose, DyPs are a promising class of biocatalysts for lignin degradation that not only represents a source of valuable fine chemicals, but it also constitutes a fundamental step in biofuels production. Research efforts are envisioned for the improvement of the activity of DyPs against lignin, through directed evolution, ration protein design, or one-pot combination with other enzymes to reach satisfactory conversion levels for industrial applications.

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 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.

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects.

Dye-decolorizing peroxidases (DyPs) are a family of H2O2-dependent heme peroxidases, which have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been uncovered for the B-class ElDyP from Enterobacter lignolyticus. Its structure revealed that a water molecule acts as the sixth axial ligand with two channels at diameters of ~3.0 and 8.0 Å leading to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs' bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type ElDyP with D2O2 at pH 3.5 suggested that cmpd 0 deprotonation by the distal aspartate is rate-limiting in the formation of cmpd I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with cmpd I instead of the free enzyme. The significant inverse sKIEs of kcat/KM and kERS* suggested that the aquo release in DyPs is mechanistically important and may explain the enzyme's adoption of two-electron reduction for cmpd I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining DyPs' acidic pH optimum. The kinetic mechanism of D143H-ElDyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs' lignolytic activity.

Factors Determining the Rate and Selectivity of 4e-/4H+ Electrocatalytic Reduction of Dioxygen by Iron Porphyrin Complexes.

Reactivity as well as selectivity are crucial in the activation and electrocatalytic reduction of molecular oxygen. Recent developments in the understanding of the mechanism of electrocatalytic O2 reduction by iron porphyrin complexes in situ using surface enhanced resonance Raman spectroscopy coupled to rotating disc electrochemistry (SERRS-RDE) in conjunction with H/D isotope effects on electrocatalytic current reveals that the rate of O2 reduction, ∼104 to 105 M-1 s-1 for simple iron porphyrins, is limited by the rate of O-O bond cleavage of an intermediate ferric peroxide species (FeIII-OOH). SERRS-RDE probes the system in operando when it is under steady state such that any intermediate species that has a greater rate of formation relative to its rate of decay, including the rate determining species, would accumulate and can be identified. This technique is particularly well suited to investigate iron porphyrin electrocatalysts as the intense symmetric ligand vibrations allow determination of the oxidation and spin states of the bound iron with high fidelity. The rate of O2 reduction could be tuned up by 3 orders of magnitude by incorporating residues in the catalyst design that can exert "push" or "pull" effects, that is, axial phenolate and thiolate ligands and distal arginine residues. Similarly the rate of O-O bond cleavage can be enhanced by several orders of magnitude upon incorporating a distal Cu site and installing the active site in a hydrophobic protein environment in synthetic models and biosynthetic protein scaffolds. The selectivity, however, is solely determined by the site of protonation of a ferric peroxide (FeIII-OOH) intermediate and can be governed by installing preorganized second sphere residues in the distal pocket. The 4e-/4H+ reduction of O2 entails protonation of the distal oxygen of the FeIII-OOH species, while 2e-/2H+ reduction requires the proximal oxygen to be protonated. Mechanistic investigations of CO2 reduction by iron porphyrins reveal that the rate-determining step is the C-O bond cleavage of a FeII-COOH species analogous to the O-O bond cleavage step of a FeIII-OOH species in O2 reduction. The selectivity, resulting in either CO or HCOOH, is determined by the site of protonation of this species. These similarities suggests that the chemical principles governing the rate and selectivity of reduction of small molecules like O2, CO2, NOx, and SOx may be quite similar in nature.

An integrated view of redox and catalytic properties of B-type PpDyP from Pseudomonas putida MET94 and its distal variants

PpDyP from Pseudomonas putida MET94 is an extremely versatile B-type dye-decolourising peroxidase (DyP) capable of efficient oxidation of a wide range of anthraquinonic and azo dyes, phenolic substrates, the non-phenolic veratryl alcohol and even manganese and ferrous ions. In reaction with H2O2 it forms a stable Compound I at a rate of (1.4±0.3)×106M−1s−1, comparable to those of classical peroxidases and other DyPs. We provide the first report of standard redox potential (E0′) of the Compound I/Native redox couple in a DyP-type peroxidase. The value of E0′Cpd I/N=1.10±0.04 (V) is similar to those found in peroxidases from the mammalian superfamily but higher than in peroxidases from the plant superfamily. Site-directed mutagenesis has been used to investigate the role of conserved distal residues, i.e. to replace aspartate 132 by asparagine, and arginine 214 and asparagine 136 by leucine. The structural, redox and catalytic properties of variants are addressed by spectroscopic, electrochemical and kinetic measurements. Our data point to the importance of the distal arginine in the catalytic mechanism of PpDyP, as also observed in DyPB from Rhodococcus jostii RHA1 but not in DyPs from the A and D subfamilies. This work reinforces the idea of existence of mechanistic variations among members of the different sub-families of DyPs with direct implications for their enzymatic properties and potential for biotechnological applications.

Acetyltransferase p300/CBP associated Factor (PCAF) regulates crosstalk-dependent acetylation of histone H3 by distal site recognition.

Epigenetic regulation is directed, in part, by the correlated placement of histone post-translational modifications, but the mechanisms controlling correlated modifications are incompletely understood. Correlations arise from crosstalk among modifications and are frequently attributed to protein–protein interactions that recruit enzymes to existing histone modifications. Here we report the use of a peptide array to discover acetyltransferase-mediated crosstalks. We show that p300/CBP associated factor (PCAF)/GCN5 activity depends on the presence of a distal arginine residue of its histone H3 substrate. Modifications to H3 Arg8 decrease PCAF acetylation of H3 Lys14, and kinetic data indicate that arginine citrullination has the strongest effect in decreasing acetylation. Mutagenesis experiments demonstrate that PCAF specifically interprets H3 Arg8 modifications through interaction with residue Tyr640 on the surface of its catalytic domain, and this interaction regulates Lys14 acetylation by substrate discrimination. PCAF discriminates modified peptides as well as semisynthetic proteins and reconstituted nucleosomes bearing Arg8 modifications. Together, this work describes a method for systematically mapping crosstalks and illustrates its application to the discovery and elucidation of novel PCAF crosstalks.

Transiently produced hypochlorite is responsible for the irreversible inhibition of chlorite dismutase.

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.

How is the distal pocket of a heme protein optimized for binding of tryptophan?

Indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase catalyze the O(2) -dependent oxidation of l-tryptophan to N-formylkynurenine. Both are heme-containing enzymes, with a proximal histidine ligand, as found in the globins and peroxidases. From the structural information available so far, the distal heme pockets of these enzymes can contain a histidine residue (in tryptophan 2,3-dioxygenases), an arginine residue and numerous hydrophobic residues that line the pocket. We have examined the functional role of each of these residues in both human indoleamine 2,3-dioxygenase and human tryptophan 2,3-dioxygenase. We found that the distal histidine does not play an essential catalytic role, although substrate binding can be affected by removing the distal arginine and reducing the hydrophobic nature of the binding pocket. We collate the information obtained in the present study with that reported in the available literature to draw comparisons across the family and to provide a more coherent picture of how the heme pocket is optimized for tryptophan binding.

Site-directed mutagenesis of manganese peroxidase from Phanerochaete chrysosporium in an in vitro expression system

An enzymatically active fungal manganese peroxidase (MnP) from Phanerochaete chrysosporium was synthesized in an in vitro coupled transcription–translation system. The synthesized MnP had the expected molecular mass (43,000 Da) and catalyzed the oxidation of 2,6-dimethoxyphenol (DMP) in the presence of hydrogen peroxide and Mn(II). A distal arginine residue (Arg 42) of the peroxide binding pocket and the potential N-glycosylation site (Asn 131) was site-directed mutagenized and corresponding mutant enzymes were also in vitro synthesized. Activities of the mutant enzymes towards 2,6-DMP were not significantly compromised although their dynamic characteristics were obviously different from the wild-type enzyme. The effect of the mutations was explained by using a computer-based three-dimensional modeling. These results demonstrated that in vitro expression of MnP provided a convenient and efficient system for characterization of fungal peroxidases.

Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H+-gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.

Substrate binding and protein conformational dynamics measured by 2D-IR vibrational echo spectroscopy.

Enzyme structural dynamics play a pivotal role in substrate binding and biological function, but the influence of substrate binding on enzyme dynamics has not been examined on fast time scales. In this work, picosecond dynamics of horseradish peroxidase (HRP) isoenzyme C in the free form and when ligated to a variety of small organic molecule substrates is studied by using 2D-IR vibrational echo spectroscopy. Carbon monoxide bound at the heme active site of HRP serves as a spectroscopic marker that is sensitive to the structural dynamics of the protein. In the free form, HRP assumes two distinct spectroscopic conformations that undergo fluctuations on a tens-of-picoseconds time scale. After substrate binding, HRP is locked into a single conformation that exhibits reduced amplitudes and slower time-scale structural dynamics. The decrease in carbon monoxide frequency fluctuations is attributed to reduced dynamic freedom of the distal histidine and the distal arginine, which are key residues in modulating substrate binding affinity. It is suggested that dynamic quenching caused by substrate binding can cause the protein to be locked into a conformation suitable for downstream steps in the enzymatic cycle of HRP.

Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants

In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at Nε position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at Nζ position). UV–visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. 1H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the p K a of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.

Crystal Structures of Chloroperoxidase with Its Bound Substrates and Complexed with Formate, Acetate, and Nitrate

Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 A. This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me2SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.