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BBA - Proteins and Proteomics (v.1824, #8)
Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays by B. Rusconi; A.C. Maranhao; J.P. Fuhrer; P. Krotee; S.H. Choi; F. Grun; T. Thireou; S.D. Dimitratos; D.F. Woods; O. Marinotti; M.F. Walter; E. Eliopoulos (pp. 947-953).
The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. ► In silico protein structure modeling and site-directed mutagenesis. ► Circular dichroism and ligand-binding assays to probe the effects of the point mutations. ► Interpretation of mutational effects on folding and ligand binding.
Keywords: Odorant Binding Protein; Anopheles gambiae; Circular dichroism spectroscopy; Fluorescence spectroscopy; Site directed mutagenesis; 3D modeling
Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays by B. Rusconi; A.C. Maranhao; J.P. Fuhrer; P. Krotee; S.H. Choi; F. Grun; T. Thireou; S.D. Dimitratos; D.F. Woods; O. Marinotti; M.F. Walter; E. Eliopoulos (pp. 947-953).
The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. ► In silico protein structure modeling and site-directed mutagenesis. ► Circular dichroism and ligand-binding assays to probe the effects of the point mutations. ► Interpretation of mutational effects on folding and ligand binding.
Keywords: Odorant Binding Protein; Anopheles gambiae; Circular dichroism spectroscopy; Fluorescence spectroscopy; Site directed mutagenesis; 3D modeling
Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris by Kimihiko Mizutani; Mayuko Toyoda; Yuichiro Otake; Soshi Yoshioka; Nobuyuki Takahashi; Bunzo Mikami (pp. 954-962).
The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and KM and Vmax values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)8 barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.► High-throughput construction of secretory protein expression system in P. pastoris ► Alpha-amylase of medaka fish was secretory expressed using the developed method. ► Purified recombinant protein exhibited starch hydrolysis activity. ► Structure of purified recombinant protein was determined by X-ray crystallography. ► Enzymatic property and overall structure are very similar to those of mammal.
Keywords: Abbreviations; OLAmy; Oryzias latipes; α-amylase; PCR; polymerase chain reaction; CBB; Coomassie Brilliant Blue; PAGE; polyacrylamide gel electrophoresis; GPCR; G protein-coupled receptor Pichia pastoris; Secretory protein; Expression system; X-ray structure
Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris by Kimihiko Mizutani; Mayuko Toyoda; Yuichiro Otake; Soshi Yoshioka; Nobuyuki Takahashi; Bunzo Mikami (pp. 954-962).
The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and KM and Vmax values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)8 barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.► High-throughput construction of secretory protein expression system in P. pastoris ► Alpha-amylase of medaka fish was secretory expressed using the developed method. ► Purified recombinant protein exhibited starch hydrolysis activity. ► Structure of purified recombinant protein was determined by X-ray crystallography. ► Enzymatic property and overall structure are very similar to those of mammal.
Keywords: Abbreviations; OLAmy; Oryzias latipes; α-amylase; PCR; polymerase chain reaction; CBB; Coomassie Brilliant Blue; PAGE; polyacrylamide gel electrophoresis; GPCR; G protein-coupled receptor Pichia pastoris; Secretory protein; Expression system; X-ray structure
Size, orientation and organization of oligomers that nucleate amyloid fibrils: Clues from MD simulations of pre-formed aggregates by Alka Srivastava; Petety V. Balaji (pp. 963-973).
All-atom MD simulations of pre-formed aggregates of GNNQQNY with variable size (5 to 16 peptides), orientation (parallel or anti-parallel), organization (single or double sheet, with or without twist), charge status of termini and temperature (300 and 330K) have been performed for 50ns each (68 simulations; total time=3.4μs). Double‐layer systems are stable irrespective of whether the peptides within the sheet are oriented parallel or anti-parallel. The lifetime of single sheet systems is determined by the protonation status, nature of association of peptides and the size of the aggregates. For example, single sheet 8-mers are stable with parallel arrangement and neutral termini, or with anti-parallel arrangement and charged termini. This suggests that the residues flanking the amyloidogenic sequence also play an important role in determining the organization of peptides in an aggregate. Twist of the cross-beta sheets is found to be intrinsic to the aggregates. Main chain H-bonds are key determinants of stability and loss of these H-bonds is followed by disorder and/or dissociation of the peptide despite the presence of side chain hydrogen bonds. Aggregates are inherently asymmetric along the fiber axis and dissociation from the C-edge is observed more often. An aggregate can disintegrate into smaller-sized oligomers or the edge peptides can dissociate sequentially. A variety of dissociation and disintegration events are observed pointing to the existence of multiple pathways for association during nucleation. It appears that a heterogeneous mixture of oligomers of different sizes exist prior to the formation of the critical nucleus.► Critical nucleus emerges from a heterogeneous mixture of oligomers. ► Asymmetry along the fiber axis determines the pattern of dissociation. ► Formation of double layer gives stability to otherwise unstable single sheets. ► Orientation of peptides in a single sheet is determined by terminal charge. ► Twist is integral to amyloid aggregates.
Keywords: Amyloid; Pre-formed aggregate; Critical nucleus; Aggregation pathway; Unstable oligomer
Size, orientation and organization of oligomers that nucleate amyloid fibrils: Clues from MD simulations of pre-formed aggregates by Alka Srivastava; Petety V. Balaji (pp. 963-973).
All-atom MD simulations of pre-formed aggregates of GNNQQNY with variable size (5 to 16 peptides), orientation (parallel or anti-parallel), organization (single or double sheet, with or without twist), charge status of termini and temperature (300 and 330K) have been performed for 50ns each (68 simulations; total time=3.4μs). Double‐layer systems are stable irrespective of whether the peptides within the sheet are oriented parallel or anti-parallel. The lifetime of single sheet systems is determined by the protonation status, nature of association of peptides and the size of the aggregates. For example, single sheet 8-mers are stable with parallel arrangement and neutral termini, or with anti-parallel arrangement and charged termini. This suggests that the residues flanking the amyloidogenic sequence also play an important role in determining the organization of peptides in an aggregate. Twist of the cross-beta sheets is found to be intrinsic to the aggregates. Main chain H-bonds are key determinants of stability and loss of these H-bonds is followed by disorder and/or dissociation of the peptide despite the presence of side chain hydrogen bonds. Aggregates are inherently asymmetric along the fiber axis and dissociation from the C-edge is observed more often. An aggregate can disintegrate into smaller-sized oligomers or the edge peptides can dissociate sequentially. A variety of dissociation and disintegration events are observed pointing to the existence of multiple pathways for association during nucleation. It appears that a heterogeneous mixture of oligomers of different sizes exist prior to the formation of the critical nucleus.► Critical nucleus emerges from a heterogeneous mixture of oligomers. ► Asymmetry along the fiber axis determines the pattern of dissociation. ► Formation of double layer gives stability to otherwise unstable single sheets. ► Orientation of peptides in a single sheet is determined by terminal charge. ► Twist is integral to amyloid aggregates.
Keywords: Amyloid; Pre-formed aggregate; Critical nucleus; Aggregation pathway; Unstable oligomer
Effects of histone acetylation and CpG methylation on the structure of nucleosomes by Ju Yeon Lee; Tae-Hee Lee (pp. 974-982).
Nucleosomes are the fundamental packing units of the eukaryotic genome. A nucleosome core particle comprises an octameric histone core wrapped around by ~147bp DNA. Histones and DNA are targets for covalent modifications mediated by various chromatin modification enzymes. These modifications play crucial roles in various gene regulation activities. A group of common hypotheses for the mechanisms of gene regulation involves changes in the structure and structural dynamics of chromatin induced by chromatin modifications. We employed single molecule fluorescence methods to test these hypotheses by monitoring the structure and structural dynamics of nucleosomes before and after histone acetylation and DNA methylation, two of the best-conserved chromatin modifications throughout eukaryotes. Our studies revealed that these modifications induce changes in the structure and structural dynamics of nucleosomes that may contribute directly to the formation of open or repressive chromatin conformation.► Physical properties of nucleosomes were studied using single molecule methods. ► Some chromatin modifications alter the physical properties of nucleosomes. ► Histone acetylation induces slight unwrapping of nucleosomal DNA. ► Histone acetylation weakens internucleosomal interactions. ► DNA methylation rigidifies nucleosomal DNA and induces compaction of nucleosomes.
Keywords: Nucleosome; Histone acetylation; DNA methylation; Single molecule; FRET; Fluorescence anisotropy
Effects of histone acetylation and CpG methylation on the structure of nucleosomes by Ju Yeon Lee; Tae-Hee Lee (pp. 974-982).
Nucleosomes are the fundamental packing units of the eukaryotic genome. A nucleosome core particle comprises an octameric histone core wrapped around by ~147bp DNA. Histones and DNA are targets for covalent modifications mediated by various chromatin modification enzymes. These modifications play crucial roles in various gene regulation activities. A group of common hypotheses for the mechanisms of gene regulation involves changes in the structure and structural dynamics of chromatin induced by chromatin modifications. We employed single molecule fluorescence methods to test these hypotheses by monitoring the structure and structural dynamics of nucleosomes before and after histone acetylation and DNA methylation, two of the best-conserved chromatin modifications throughout eukaryotes. Our studies revealed that these modifications induce changes in the structure and structural dynamics of nucleosomes that may contribute directly to the formation of open or repressive chromatin conformation.► Physical properties of nucleosomes were studied using single molecule methods. ► Some chromatin modifications alter the physical properties of nucleosomes. ► Histone acetylation induces slight unwrapping of nucleosomal DNA. ► Histone acetylation weakens internucleosomal interactions. ► DNA methylation rigidifies nucleosomal DNA and induces compaction of nucleosomes.
Keywords: Nucleosome; Histone acetylation; DNA methylation; Single molecule; FRET; Fluorescence anisotropy
Inter-domain interactions influence the stability and catalytic activity of the bi-domain protein tyrosine phosphatase PTP99A by Lalima L. Madan; Kapil Goutam; B. Gopal (pp. 983-990).
The two protein tyrosine phosphatase (PTP) domains in bi-domain PTPs share high sequence and structural similarity. However, only one of the two PTP domains is catalytically active. Here we describe biochemical studies on the two tandem PTP domains of the bi-domain PTP, PTP99A. Phosphatase activity, monitored using small molecule as well as peptide substrates, revealed that the inactive (D2) domain activates the catalytic (D1) domain. Thermodynamic measurements suggest that the inactive D2 domain stabilizes the bi-domain (D1–D2) protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by few interactions at the inter-domain interface. In particular, mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent location to the so-called allosteric site of the canonical single domain PTP, PTP1B. These observations suggest functional optimization in bi-domain PTPs whereby the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme.Display Omitted► The functional role of silent PTP domains in bi-domain phosphatases was examined. ► The silent PTP domain acts as a non-essential partially mixed activator. ► Conserved residues at the domain interface modulate inter-domain interactions. ► Mutation of a basic residue at the interface abrogates domain-domain interaction.
Keywords: Silent domain; Catalytic activity; Activation; Phosphatase; Allosteric site; Domain–domain interaction
Inter-domain interactions influence the stability and catalytic activity of the bi-domain protein tyrosine phosphatase PTP99A by Lalima L. Madan; Kapil Goutam; B. Gopal (pp. 983-990).
The two protein tyrosine phosphatase (PTP) domains in bi-domain PTPs share high sequence and structural similarity. However, only one of the two PTP domains is catalytically active. Here we describe biochemical studies on the two tandem PTP domains of the bi-domain PTP, PTP99A. Phosphatase activity, monitored using small molecule as well as peptide substrates, revealed that the inactive (D2) domain activates the catalytic (D1) domain. Thermodynamic measurements suggest that the inactive D2 domain stabilizes the bi-domain (D1–D2) protein. The mechanism by which the D2 domain activates and stabilizes the bi-domain protein is governed by few interactions at the inter-domain interface. In particular, mutating Lys990 at the interface attenuates inter-domain communication. This residue is located at a structurally equivalent location to the so-called allosteric site of the canonical single domain PTP, PTP1B. These observations suggest functional optimization in bi-domain PTPs whereby the inactive PTP domain modulates the catalytic activity of the bi-domain enzyme.Display Omitted► The functional role of silent PTP domains in bi-domain phosphatases was examined. ► The silent PTP domain acts as a non-essential partially mixed activator. ► Conserved residues at the domain interface modulate inter-domain interactions. ► Mutation of a basic residue at the interface abrogates domain-domain interaction.
Keywords: Silent domain; Catalytic activity; Activation; Phosphatase; Allosteric site; Domain–domain interaction
Non-canonical residues of the marginally stable monomeric ubiquitin conjugase from goldfish are involved in binding to the C terminus of Ring 1B by David Aguado-Llera; Domenech Rosa Doménech; Marco Marenchino; Miguel Vidal; José L. Neira (pp. 991-1001).
E2 ubiquitin conjugases are ~20kDa enzymes involved in ubiquitination processes in eukaryotes. The E2s are responsible for the transference of ubiquitin (Ub) to E3 enzymes, which finally transfer Ub to diverse target proteins, labelling them for degradation, localization and regulation. Although their functions are relatively well-characterized, their conformational stabilities are poorly known. In this work, we have used, as a model for our biophysical and binding studies, the E2-C from Carassius auratus (goldfish), a homologue of the human ubiquitin conjugase UbcH10. E2-Cca was a monomeric protein with an elongated shape; moreover, the protein was only marginally stable within a narrow pH range (from 6.0 to 8.0). We also explored the binding of E2-Cca towards non-canonical E3 ligases. Binding of E2-Cca to the C terminus of murine Ring 1B (C-Ring1B), which does not contain the RING finger of the whole Ring1B, occurred with an affinity of ~400nM, as shown by fluorescence and ITC. Furthermore, binding of E2-Cca to C-Ring1B did not occur at its canonical E2-loops, since residues M43 and F53, far away from those loops, were involved in binding. Thus, the C-Ring1B-interacting region of E2-Cca comprises the first β-strand and nearby residues.Display Omitted► The conformational stability of E2-Cca is very low. ► The C-terminal region of Ring1B, C-Ring1B, binds E2-Cca, with a 400 nM affinity. ► The E2-Cca-binding region of C-Ring1B does not involve any RING finger. ► The C-Ring1B-binding region of E2-Cca does not involve the canonical loops. ► Residues M43 and F53 of E2-Cca are critical in the binding of both proteins.
Keywords: Abbreviations; ANS; 1-anilino-8-naphtalene sulfonate; AUC; analytical ultracentrifugation; C-Ring 1B; the C terminus of murine Ring 1B, comprising residues 227–334 of the intact Ring 1B; CD; circular dichroism; DOSY; diffusion ordered spectroscopy; DSC; differential scanning calorimetry; E1; ubiquitin-activating enzyme; E2; ubiquitin-conjugating enzyme; E3; ubiquitin-ligase enzyme; E2-C; ca; E2-C from; Carassius auratus; (goldfish); E6-AP; E6-associated protein; HECT; homologous with E6-AP C terminus; ITC; isothermal titration calorimetry; R; hydrodynamic radius; RING; really interesting new gene; SEC; size exclusion chromatography; SUMO; small ubiquitin-related modifier; T; m; the thermal-denaturation midpoint of fluorescence and CD thermal denaturations; Ub; ubiquitinFolding; Stability; Binding; Ubiquitination; Ring proteins; Circular dichroism
Non-canonical residues of the marginally stable monomeric ubiquitin conjugase from goldfish are involved in binding to the C terminus of Ring 1B by David Aguado-Llera; Domenech Rosa Doménech; Marco Marenchino; Miguel Vidal; José L. Neira (pp. 991-1001).
E2 ubiquitin conjugases are ~20kDa enzymes involved in ubiquitination processes in eukaryotes. The E2s are responsible for the transference of ubiquitin (Ub) to E3 enzymes, which finally transfer Ub to diverse target proteins, labelling them for degradation, localization and regulation. Although their functions are relatively well-characterized, their conformational stabilities are poorly known. In this work, we have used, as a model for our biophysical and binding studies, the E2-C from Carassius auratus (goldfish), a homologue of the human ubiquitin conjugase UbcH10. E2-Cca was a monomeric protein with an elongated shape; moreover, the protein was only marginally stable within a narrow pH range (from 6.0 to 8.0). We also explored the binding of E2-Cca towards non-canonical E3 ligases. Binding of E2-Cca to the C terminus of murine Ring 1B (C-Ring1B), which does not contain the RING finger of the whole Ring1B, occurred with an affinity of ~400nM, as shown by fluorescence and ITC. Furthermore, binding of E2-Cca to C-Ring1B did not occur at its canonical E2-loops, since residues M43 and F53, far away from those loops, were involved in binding. Thus, the C-Ring1B-interacting region of E2-Cca comprises the first β-strand and nearby residues.Display Omitted► The conformational stability of E2-Cca is very low. ► The C-terminal region of Ring1B, C-Ring1B, binds E2-Cca, with a 400 nM affinity. ► The E2-Cca-binding region of C-Ring1B does not involve any RING finger. ► The C-Ring1B-binding region of E2-Cca does not involve the canonical loops. ► Residues M43 and F53 of E2-Cca are critical in the binding of both proteins.
Keywords: Abbreviations; ANS; 1-anilino-8-naphtalene sulfonate; AUC; analytical ultracentrifugation; C-Ring 1B; the C terminus of murine Ring 1B, comprising residues 227–334 of the intact Ring 1B; CD; circular dichroism; DOSY; diffusion ordered spectroscopy; DSC; differential scanning calorimetry; E1; ubiquitin-activating enzyme; E2; ubiquitin-conjugating enzyme; E3; ubiquitin-ligase enzyme; E2-C; ca; E2-C from; Carassius auratus; (goldfish); E6-AP; E6-associated protein; HECT; homologous with E6-AP C terminus; ITC; isothermal titration calorimetry; R; hydrodynamic radius; RING; really interesting new gene; SEC; size exclusion chromatography; SUMO; small ubiquitin-related modifier; T; m; the thermal-denaturation midpoint of fluorescence and CD thermal denaturations; Ub; ubiquitinFolding; Stability; Binding; Ubiquitination; Ring proteins; Circular dichroism
Role of K+ binding residues in stabilization of heme spin state of Leishmania major peroxidase by Swati Pal; Rajesh K. Yadav; Subrata Adak (pp. 1002-1007).
The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K+ site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV–visible spectrophotometry, we show that the removal of K+ binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K+ site double mutants in the presence of H2O2. Similarly electron donor oxidation rate was two orders less for the K+ site double mutants compared to the wild type. These data show that K+ functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP.► Leishmania major peroxidase is structurally hybrid of APX and CCP. ► We analyzed the function of proximal K+ binding residues in this protein. ► The catalytic efficiency in mutant was decreased by about 100 times. ► Proximal K+ binding is important for preserving the spin state of heme iron.
Keywords: Abbreviations; APX; plant ascorbate peroxidase; LmP; Leishmania major; peroxidase; CCP; yeast cytochrome; c; peroxidase; HS; high spin; LS; low spin; WT; wild type Leishmania; Heme protein; Peroxidase; Steady-state catalysis; Rapid kinetics and mutation
Role of K+ binding residues in stabilization of heme spin state of Leishmania major peroxidase by Swati Pal; Rajesh K. Yadav; Subrata Adak (pp. 1002-1007).
The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K+ site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV–visible spectrophotometry, we show that the removal of K+ binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K+ site double mutants in the presence of H2O2. Similarly electron donor oxidation rate was two orders less for the K+ site double mutants compared to the wild type. These data show that K+ functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP.► Leishmania major peroxidase is structurally hybrid of APX and CCP. ► We analyzed the function of proximal K+ binding residues in this protein. ► The catalytic efficiency in mutant was decreased by about 100 times. ► Proximal K+ binding is important for preserving the spin state of heme iron.
Keywords: Abbreviations; APX; plant ascorbate peroxidase; LmP; Leishmania major; peroxidase; CCP; yeast cytochrome; c; peroxidase; HS; high spin; LS; low spin; WT; wild type Leishmania; Heme protein; Peroxidase; Steady-state catalysis; Rapid kinetics and mutation