Distal Histidine Residue Research Articles

Structure and non-reactive dynamics of the dimeric catalytic domain of human carbonic anhydrase IX

Despite the availability of high resolution crystal structure of the catalytic domain of human carbonic anhydrase IX (denoted as HCA IX-c), its solution structure and functional dynamics are mostly unknown. In this article, we report classical molecular dynamics (MD) simulation studies on the structure and non-reactive dynamics of dimeric HCA IX-c (d-HCA IX-c, with chains A and B) in water and compare them to each chain simulated in water as a monomeric solute (mA/mB). While the average solution structures appear to be largely similar in all systems, d-HCA IX-c shows significant deviations from mA and mB in terms of non-reactive reorganization of the active site needed to initiate the rate determining proton transfer step between a zinc-bound water and one of the sidechain N-atoms of a distal histidine residue (His-64). First, 2−6 water molecules are found to form fluctuating hydrogen bonded networks that would serve as proton paths across the active site. A minimally frustrated core of each monomer in the dimeric protein is found to be associated with a significantly higher likelihood of forming complete proton transfer pathways mediated by 2 water molecules synchronously at the two active sites hosted by the monomeric chains. The relative stability and kinetics of transition between two major sidechain rotamers of His-64 are also affected by dimerization. The sidechain rotation of His-64 is found to involve at least one transition as slow as 105s−1 in chain A of d-HCA IX-c thereby rendering it slower than the actual rate determining proton transfer step. This observation indicates an important deviation from the widely accepted mechanism of catalysis by HCAs.

Hb H disease associated with compound heterozygosity for --SEA deletion and a novel alpha globin chain variant (HBA2:c.175C>A) on the distal histidine in a Chinese family

ABSTRACT Objectives: In clinical practice, the majority of α-thalassaemia cases arise from deletions of the α-globin genes. However, a subset of cases is attributed to rare haemoglobin variants, which can manifest with borderline or normal screening results, potentially leading to missed diagnoses in clinical practice. Methods: Blood samples were collected from family members and underwent haematological, DNA and RNA analysis. Results: The five-month-old proband presented a haematological phenotype consistent with Hb H disease. The mother’s haematology profile was consistent with an α-thalassaemia carrier, while the father exhibited a borderline reduction in MCV and MCH. MALDI-TOF identified an abnormal α-chain in the proband. DNA analysis revealed a novel α-globin variant (HBA2:c.175C>A, α58His>Asn, Hb DG-Nancheng) affecting the distal histidine in the family. The father and the mother had α-genotype of --SEA/αα and αDG-Nanchengα/αα, respectively; while the proband inherited both mutant alleles (--SEA/αDG-Nanchengα). Sequencing of cDNA from HBA2 gene identified an equal ratio of normal and mutant alleles. Conclusion: This rare case highlighted the importance of identifying rare haemoglobin variant during prenatal screening. The clinical and genetic data provides useful information on the pathogenicity of this variant and further insight into the role of distal histidine residue of α-globin.

A robust and facile label-free method for highly sensitive colorimetric detection of ascorbic acid in fresh fruits based on peroxidase-like activity of modified FeCo-LDH@WO3 nanocomposite

Many compounds such as amino acids and oligonucleotides have been shown to effectively change peroxidase-like activity of nanoparticles. While a few studies have focused on mimicking the active site of natural enzymes on nanozymes and thus increasing their substrate affinity. Therefore, in this work, the surface of FeCo@WO3 nanocomposite was modified using guanosine triphosphate (GTP) to mimic the histidine of peroxidase enzyme's active site and its modification was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FT-IR). Then, the peroxidase-mimicking activity of the modified nanocomposite was tested using a colorimetric method, based on the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). It was found that GTP improves the activity of FeCo@WO3 as a natural peroxidase active site's distal histidine residue. Ascorbic acid (AA) is a powerful antioxidant that induces the reduction of blue color (oxidized TMB) ox-TMB to colorless TMB. The colorimetric method was applied for the sensitive detection of AA in common fruits. The linear range of AA was 10–100 μM with a limit of detection (LOD) of 0.27 μM, which provides a rapid and sensitive method for testing AA in the field of food analysis.

Secondary Coordination Sphere Influences the Formation of Fe(III)-O or Fe(III)-OH in Nitrite Reduction: A Synthetic and Computational Study.

The reduction of nitrite (NO2-) to generate nitric oxide (NO) is a significant area of research due to their roles in the global nitrogen cycle. Here, we describe various modifications of the tris(5-cyclohexyliminopyrrol-2-ylmethyl)amine H3[N(piR)3] ligand where the steric bulk and acidity of the secondary coordination sphere were explored in the non-heme iron system for nitrite reduction. The cyclohexyl and 2,4,6-trimethylphenyl variants of the ligand were used to probe the mechanism of nitrite reduction. While previously stoichiometric addition of nitrite to the iron(II)-species generated an iron(III)-oxo complex, changing the secondary coordination sphere to mesityl resulted in an iron(III)-hydroxo complex. Subsequent addition of an electron and two protons led to the release of water and regeneration of the starting iron(II) catalyst. This sequence mirrored the proposed mechanism of nitrite reduction in biological systems, where the distal histidine residue shuttles protons to the active site. Computational studies aimed at interrogating the dissimilar behavior of the cyclohexyl and mesityl ligand systems resulting in Fe(III)-oxo and Fe(III)-hydroxo complexes, respectively, shed light on the key role of H-bonds involving the secondary coordination sphere in the relative stability of these species.

Myoglobin ligand gate mechanism analysis by a novel 3D visualization technique

A protein is commonly visualized as a discrete piecewise linear curve, conventionally characterized in terms of the extrinsically determined Ramachandran angles. However, in addition to the extrinsic geometry, the protein has also two independent intrinsic geometric structures, determined by the peptide planes and the side chains respectively. Here we develop a novel 3D visualization method that instead of the extrinsic geometry utilizes the intrinsic geometry of side chains. We base our approach on a series of orthonormal coordinate frames along the protein side chains in combination with a mapping of the atoms positions onto a unit sphere, for visualization purposes. We develop our methodology in terms of an example, by analyzing the acidity dependence of the presumed myoglobin ligand gate. In the literature, the ligand gate is often asserted to be highly localized at the distal histidine, its functioning being regulated by environmental changes. Thus, we investigate whether any $${\mathrm{pH}}$$ dependence can be detected in the orientation of the distal and proximal histidine residues, using existing crystallographic data. We observe no $${\mathrm{pH}}$$ dependence, in support of the alternative proposals that the ligand gate is more complex and might even be located elsewhere. Our methodology should help the planning of future myoglobin structure experiments, to identify the ligand gate position and its mechanism. More generally, our methodology is designed to visually depict the spatial orientation of side chain covalent bonds in a protein. As such, it can be eventually advanced into a general visual 3D tool for protein structure analysis for purposes of prediction, validation and refinement. It can serve as a complement to widely used visualization suites such as VMD, Jmol, PyMOL and others.

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin

Hydrogen sulfide is a critical signaling molecule, but high concentrations cause cellular toxicity. A four-enzyme pathway in the mitochondrion detoxifies H2S by converting it to thiosulfate and sulfate. Recent studies have shown that globins like hemoglobin and myoglobin can also oxidize H2S to thiosulfate and hydropolysulfides. Neuroglobin, a globin enriched in the brain, was reported to bind H2S tightly and was postulated to play a role in modulating neuronal sensitivity to H2S in conditions such as stroke. However, the H2S reactivity of the coordinately saturated heme in neuroglobin is expected a priori to be substantially lower than that of the 5-coordinate hemes present in myoglobin and hemoglobin. To resolve this discrepancy, we explored the role of the distal histidine residue in muting the reactivity of human neuroglobin toward H2S. Ferric neuroglobin is slowly reduced by H2S and catalyzes its inefficient oxidative conversion to thiosulfate. Mutation of the distal His64 residue to alanine promotes rapid binding of H2S and its efficient conversion to oxidized products. X-ray absorption, EPR, and resonance Raman spectroscopy highlight the chemically different reaction options influenced by the distal histidine ligand. This study provides mechanistic insights into how the distal heme ligand in neuroglobin caps its reactivity toward H2S and identifies by cryo-mass spectrometry a range of sulfide oxidation products with 2-6 catenated sulfur atoms with or without oxygen insertion, which accumulate in the absence of the His64 ligand.

Amplified Peroxidase-Like Activity in Iron Oxide Nanoparticles Using Adenosine Monophosphate: Application to Urinary Protein Sensing.

Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe3O4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe3O4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H2O2) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe3O4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe2+/Fe3+ sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H2O2. Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe3O4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe3O4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe3O4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe3O4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe3O4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.

Unveiling the basis of alkaline stability of an evolved versatile peroxidase.

A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP (versatile peroxidase) expressed in Saccharomyces cerevisiae [García-Ruiz, González-Pérez, Ruiz-Dueñas, Martínez and Alcalde (2012) Biochem. J. 441: , 487-498]. 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120h of incubation at pH9 compared with complete inactivation of the native enzyme after only 1h). The latter is highly demanded for biorefinery applications. In the present study we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were expressed in Escherichia coli, and their alkaline stability and biochemical properties were determined. In addition, the crystal structures of 2-1B and one of the intermediate variants were solved and carefully analysed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections between haem and helix B (where the distal histidine residue is located), and formation of new electrostatic interactions, that avoided the hexa-co-ordination of the haem iron. These new structural determinants stabilized the haem and its environment, helping to maintain the structural enzyme integrity (with penta-co-ordinated haem iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305in the proximal side, prompted by the Q202L mutation, further enhanced the stability.

Mutational Analysis of the Catalytic Mechanism of a Peroxidase Enzyme from the Diatom Thalassiosira pseudonana

Heme iron is integral to numerous biochemical processes including electron transfer, oxygen transport, and catalysis of oxidation‐reduction reactions. Given that iron is only sparingly soluble in water, enzymes that contain iron in marine organisms must play an important physiological role. We have identified a putative heme‐containing enzyme, Tp21683, in the published genome of the marine diatom Thalassiosira pseudonana. Tp21683 has a C‐terminal predicted b‐type heme peroxidase domain and an N‐terminal cysteine‐rich domain of unknown function. We hypothesize that the role of Tp21683 is to relieve oxidative stress, a biological process critical to survival of photosynthetic organisms including diatoms. Here, we report the recombinant expression of the C‐terminal peroxidase domain of Tp21683 in Escherichia coli and successful refolding and purification to greater than 80% purity. Preliminary data indicate that Tp21683 turns over hydrogen peroxide in the presence of the small molecule electron donors ABTS and guaiacol. To confirm that the peroxidase activity of Tp21683 proceeds by a mechanism similar to other b‐type heme peroxidases, we constructed two separate site‐directed mutants at key amino acids in the predicted active site of Tp21683. We hypothesize that replacing the distal histidine residue, His221, with alanine will result in severe deficiencies in hydrogen peroxide turnover rate because a key proton donor and acceptor has been removed from the active site. Mutation of a conserved tryptophan located near the proximal histidine, Trp365, with a phenylalanine residue should highlight whether Tp21683 forms a protein‐based cation radical or a porphyrin‐based cation radical during the first step of hydrogen peroxide turnover. If Tp21683 uses a protein‐based radical, like the model enzyme yeast cytochrome c peroxidase, the W365F mutation will lead to a dramatic decrease in turnover rate because the enzyme is unable to form a stable compound I. Tp21683 mutants H221A and W365F will be recombinantly expressed and purified similarly to the wild‐type enzyme and the kinetic parameters will be assessed in the presence of hydrogen peroxide and ABTS. Verifying the roles of histidine 221 and tryptophan 365 in Tp21683 will further support our assignment of this previously uncharacterized enzyme as a heme peroxidase.

Regulating the coordination state of a heme protein by a designed distal hydrogen-bonding network.

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.

Electronic Control of Discrimination between O2 and CO in Myoglobin Lacking the Distal Histidine Residue

We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties of the H64L mutant of myoglobin reconstituted with chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities, in order to elucidate the effect of the removal of the distal His64 on the control of both the O2 affinity and discrimination between O2 and CO of the protein by the intrinsic heme Fe reactivity through the electron density of the heme Fe atom (ρFe). The study revealed that, as in the case of the native protein, the O2 affinity of the H64L mutant protein is regulated by the ρFe value in such a manner that the O2 affinity of the protein decreases, due to an increase in the O2 dissociation rate constant, with a decrease in the ρFe value, and that the O2 affinities of the mutant and native proteins are affected comparably by a given change in the ρFe value. On the other hand, the CO affinity of the H64L mutant protein was found to increase, due to a decrease in the CO dissociation rate constant, with a decrease in the ρFe value, whereas that of the native protein was essentially independent of a change in the ρFe value. As a result, the regulation of the O2/CO discrimination in the protein through the ρFe value is affected by the distal His64. Thus, the study revealed that the electronic tuning of the intrinsic heme Fe reactivity through the ρFe value plays a vital role in the regulation of the protein function, as the heme environment furnished by the distal His64 does.

Clarification of the role of protein in carbonmonoxy myoglobin by investigating electronic states

This article reports the proton tautomerization effects of distal histidine residues in carbonmonoxy myoglobin according to the density functional calculations of the whole protein. The electron eigenstates and electrostatic potential (ESP) distributed around heme and its pocket vary significantly depending on the protonation positions of the distal histidine residues. To investigate the range over which the electronic structures are affected by the proton tautomerization, the quantum mechanics/molecular mechanics (QM/MM) method is applied to probe the QM size to reproduce the atomic partial charges and ESP around the active center. Consequently, we show that these properties converged for the 300 pm QM/MM system in this study. During the analysis, we also find that amino residues such as Phe43, Val68, and Phe138 interact strongly with heme through orbital mixing, indicating that the protein is a medium not only interacting with the reaction center, but also buffering on electrons. © 2013 Wiley Periodicals, Inc.

Probing the conformational mobility of the active site of a heme peroxidase

We have previously demonstrated (Badyal et al., J. Biol. Chem., 2006, 281, 24512) that removal of the active site tryptophan (Trp41) in ascorbate peroxidase increases the conformational mobility of the distal histidine residue (His42) and that His42 coordinates to the iron in the oxidised W41A enzyme to give a 6-coordinate, low-spin peroxidase. In this work, we probe the conformational flexibility of the active site in more detail. We examine whether other residues (Cys, Tyr, Met) can also ligate to the heme at position 42; we find that introduction of other ligating amino acids created a cavity in the heme pocket, but that formation of 6-coordinate heme is not observed. In addition, we examine the role of Asn-71, which hydrogen bonds to His42 and tethers the distal histidine in the active site pocket; we find that removal of this hydrogen bond increases the proportion of low-spin heme. We suggest that, in addition to its well-known role in facilitating the reaction with peroxide, His42 also plays a role in defining the shape and folding of the active site pocket.

Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System

Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme c-type cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an ~35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.

Chemical Rescue of the Distal Histidine Mutants of Tryptophan 2,3-Dioxygenase

Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.

Amino acid sequence of myoglobin from white-tailed deer (Odocoileus virginianus)

Our objective was to determine the primary structure of white-tailed deer myoglobin (Mb). White-tailed deer Mb was isolated from cardiac muscles employing ammonium sulfate precipitation and gel-filtration chromatography. The amino acid sequence was determined by Edman degradation. Sequence analyses of intact Mb as well as tryptic- and cyanogen bromide-peptides yielded the complete primary structure of white-tailed deer Mb, which shared 100% similarity with red deer Mb. White-tailed deer Mb consists of 153 amino acid residues and shares more than 96% sequence similarity with myoglobins from meat-producing ruminants, such as cattle, buffalo, sheep, and goat. Similar to sheep and goat myoglobins, white-tailed deer Mb contains 12 histidine residues. Proximal (position 93) and distal (position 64) histidine residues responsible for maintaining the stability of heme are conserved in white-tailed deer Mb.

New Roles Assigned to the α1–β1 (and α2–β2) Interface of the Human Hemoglobin Molecule from Physiological to Cellular

Cellular life is reliant upon rapid and efficient responses to internal and external conditions. The basic molecular events associated with these processes are the structural transitions of the proteins (structural protein allostery) involved. From this view, the human hemoglobin (Hb) molecule (α2β2) holds a special position in this field. Hb has two types of αβ interface (i.e., α1β1 [and α2β2] and α1β2 [and α2β1]). The latter α1–β2 (and α2–β1) interface is known to be associated with cooperative O2 binding, and exhibits principal roles if the molecule goes from its deoxy to oxy quaternary structure. However, the role of the former α1–β1 (and α2–β2) interface has been unclear for a long time. In this regard, important and intriguing observations have been accumulating. A new role was attributed first as stabilizing the HbO2 tetramer against acidic autoxidation. That is, the α1–β1 (and α2–β2) interface produces a conformational constraint in the β chain whereby the distal (E7) histidine (His) residue is tilted slightly away from the bound O2 so as to prevent proton-catalyzed displacement of O2– by a solvent water molecule. The β chains thus acquire pH-dependent delayed autoxidation in the HbO2 tetramer. The next role was suggested by our studies searching for similar phenomena in normal human erythrocytes under mild heating. Tilting of the distal (E7) His in turn triggered degradation of the Hb molecule to hemichrome, and subsequent clustering of Heinz bodies within the erythrocyte. As Heinz body-containing red cells become trapped in the spleen, it was demonstrated that the α1–β1 (and α2–β2) interface may exert delicate control of the fate (removal) of its own erythrocyte. Herein we review and summarize the related results and current interpretation of the oxidative behavior of human Hb, emphasizing the correlation between hemichrome emergence and Heinz-body formation, and specifically discuss the new roles assigned to the α1–β1 (and α2–β2) interface. The α1–β1 (and α2–β2) interface seems to adequately differentiate between the two types of function (dual roles) from physiological to cellular.

Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using “Peroxidase-like” Myoglobin Mutants and “Myoglobin-like” DHP Mutants

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ∼10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ∼0.3 and ∼0.8 Å, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.

Theoretical study of the mechanism of oxoiron(IV) formation from H2O2 and a nonheme iron(II) complex: O-O cleavage involving proton-coupled electron transfer.

It has recently been shown that the nonheme oxoiron(IV) species supported by the 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane ligand (TMC) can be generated in near-quantitative yield by reacting [Fe(II)(TMC)(OTf)(2)] with a stoichiometric amount of H(2)O(2) in CH(3)CN in the presence of 2,6-lutidine (Li, F.; England, J.; Que, L., Jr. J. Am. Chem. Soc. 2010, 132, 2134-2135). This finding has major implications for O-O bond cleavage events in both Fenton chemistry and nonheme iron enzymes. To understand the mechanism of this process, especially the intimate details of the O-O bond cleavage step, a series of density functional theory (DFT) calculations and analyses have been carried out. Two distinct reaction paths (A and B) were identified. Path A consists of two principal steps: (1) coordination of H(2)O(2) to Fe(II) and (2) a combination of partial homolytic O-O bond cleavage and proton-coupled electron transfer (PCET). The latter combination renders the rate-limiting O-O cleavage effectively a heterolytic process. Path B proceeds via a simultaneous homolytic O-O bond cleavage of H(2)O(2) and Fe-O bond formation. This is followed by H abstraction from the resultant Fe(III)-OH species by an •OH radical. Calculations suggest that path B is plausible in the absence of base. However, once 2,6-lutidine is added to the reacting system, the reaction barrier is lowered and more importantly the mechanistic path switches to path A, where 2,6-lutidine plays an essential role as an acid-base catalyst in a manner similar to how the distal histidine or glutamate residue assists in compound I formation in heme peroxidases. The reaction was found to proceed predominantly on the quintet spin state surface, and a transition to the triplet state, the experimentally known ground state for the TMC-oxoiron(IV) species, occurs in the last stage of the oxoiron(IV) formation process.

Primary structure of turkey myoglobin

Our objective was to determine the amino acid sequence of turkey myoglobin. Turkey myoglobin was isolated from cardiac muscles via ammonium sulphate precipitation and gel-filtration chromatography. Purified turkey myoglobin, separated as a 17 kDa band in SDS–PAGE, was subjected to digestion with trypsin or aspartic acid endopeptidase. The resulting peptides were separated by reverse-phase HPLC, and then subjected to Edman degradation to obtain the amino acid sequence. The complete amino acid sequence of turkey myoglobin was determined and compared with that of poultry and red meat myoglobins. Turkey myoglobin has 153 amino acids and nine histidine residues. Proximal (position 93) and distal (position 64) histidine residues, responsible for maintaining the stability of haeme, are conserved in turkey myoglobin. Turkey myoglobin shares 100% sequence similarity with chicken myoglobin, whereas it shares 92.5% homology with ostrich, 76.5% with pig, and less than 73% with ruminant myoglobins.