Structural design of the active site for covalent attachment of the heme to the protein matrix: studies on a thermostable cytochrome P450

The molecular basis of the post-translational modification involving covalent attachment of the heme with a glutamic acid observed in some enzymes of the CYP4 family of heme monooxygenases has been investigated using site-directed mutagenesis of CYP175A1 from Thermus thermophilus. Earlier studies of CYP4 as well as the G248E mutant of CYP101A1 showed covalent linkage of the heme to a conserved glutamic acid of helix I. We have introduced Glu/Asp at the Leu80 position in the β-turn of CYP175A1, on the basis of molecular modeling studies, to assess whether formation of such a covalent linkage is limited only to helix I or whether such modification may also take place with the residue that is spatially located at a position appropriate for activation by the heme peroxidase reaction. Tandem mass spectrometry analyses of the tryptic digest of the wild type and mutants of CYP175A1 were conducted to identify any heme-bound peptide. Tryptic digestion of the L80E mutant of CYP175A1 preincubated with H(2)O(2) showed formation of GLE(-heme)TDWGESWKEARK supporting covalent linkage of Glu80 with the heme in the mutant enzyme. No such heme-bound peptides were found if the sample was not preincubated in H(2)O(2), indicating no activation of the Glu by the heme peroxidase reaction, as proposed earlier. The wild type or L80D mutant of the enzyme did not give any heme-bound peptide. Thus, the results support the idea that covalent attachment of the heme to an amino acid in the protein matrix depends on the structural design of the active site.     

Construction and engineering of a thermostable self-sufficient cytochrome P450

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP⁺ reductase (FNR): H₂N-CYP175A1-Fdx-FNR-COOH (175FR) and H₂N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V_max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k_m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 °C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.     

Purification and characterization of two extracellular peroxidases from Streptomyces sp. strain AM2, a decolorizing actinomycetes responsible for the biodegradation of natural humic acids

Two extracellular humic acids peroxidases called HaP1 and HaP2 were isolated from the Streptomyces sp. strain AM2 and, based on MALDI-TOF MS analysis. The purified enzymes were determined as monomers with molecular masses of 40,351.11 and 25,175.19 Da, respectively. The N-terminal amino acid sequences of HaP1 and HaP2 were identified, and their optimum pH values were determined as 6 and 7.5, respectively. Standard 2,4-dichlorophenol (2,4-DCP) assays showed that both enzymes had maximal activity at 55 °C. HaP2 was stable at 55 °C for more than 24 h and had a half-life of 90 min at 65 °C. Although the catalytic properties of HaP1 and HaP2 were nearly identical, their stabilities and Reinheitzahl (RZ) values were substantially different. Both peroxidases were found to be heme proteins that catalyzed the oxidation of a wide range of substrates in the presence of hydrogen peroxide (H₂O₂), with HaP2 exhibiting a broader range of substrate specificity. The characterization of peroxidase activity revealed activity against humic acids, guiacol, 2,4-DCP, l-3,4-dihydroxyphenylalanine, and 2,4,5-trichlorophenol as well as other chlorophenols in the presence of H₂O₂. However, the inhibition of peroxidase activity by the addition of potassium cyanide and sodium azide also indicated the presence of heme components in the tertiary structure of these enzymes.     

The ferrous–dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: The role of proximal histidine on its stability

Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O₂ dependent cAMP signaling (Sen Santara, et. al. Proc. Natl. Acad. Sci. U.S.A. 110, 16790–16795 (2013)). The heme iron of HemAC-Lm is expected to participate in oxygen binding and activates adenylate cyclase activity during catalysis, but its interactions with O₂ are uncharacterized. We have utilized the HemAC-Lm and stopped-flow methods to study the formation and decay of the HemAC-Lm oxygenated complex at 25 °C. Mixing of the ferrous HemAC-Lm with air-saturated buffer generates a very stable oxygenated complex with absorption maxima at 414, 540 and 576 nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O₂ at a rate of ~ 10 s^− 1. To prepare apoprotein of heme iron in HemAC-Lm, we have mutated the proximal His161 to Ala and characterized the mutant protein. The apo as well as heme reconstituted ferric state of the mutant protein shows a ~ 30 fold lower catalytic activity compared to oxygenated form of wild type protein. The oxygenated form of heme reconstituted mutant protein is highly unstable (decay rate = 6.1 s^− 1). Decomposition of the oxygenated intermediate is independent of O₂ concentration and is monophasic. Thus, the stabilization of ferrous-oxy species is an essential requirement in the wild type HemAC-Lm for a conformational alteration in the sensor domain that, sequentially, activates the adenylate cyclase domain, resulting in the synthesis of cAMP.     

Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement

A simple bio-conjugation procedure to surround a single horseradish peroxidase (HRP) enzyme molecule with dendritic polyester macromolecules (polyester-32-hydroxyl-1-carboxyl bis-MPA dendron, generation 5) was proposed. The characterization of resultant nanoparticles entitled HRP dendrozyme, was performed by transmission electron microscopy, dynamic light scattering, gel permeation chromatography and Fourier transform infrared spectroscopy. The results showed that HRP nanoparticles were spherical in shape and have an average size of 14 ± 2 nm in diameter. Furthermore, bio-conformational characterization of HRP dendrozyme was performed by means of circular dichroism and fluorescence spectroscopy to evaluate the secondary and tertiary structure changes after enzyme modification. These investigations revealed that protein conformation had small changes (in secondary and tertiary structures) after bio-conjugation. We also reported here that dendritic modification did not significantly affect the kinetic parameters of free HRP. The stabilization of HRP with dendron macromolecules as single enzyme nanoparticles resulted in improvement of half-life over 70 days storage at 4 °C as well as its tolerance under different elevated temperatures up to 80 °C and in the presence of organic solvents for 15 min. These significant results promise extensive applications of HRP particularly in harsh environmental conditions.     

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH₂ or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.     

Rational design of Bacillus stearothermophilus US100 l-arabinose isomerase: Potential applications for d-tagatose production

l-Arabinose isomerases catalyze the bioconversion of d-galactose into d-tagatose. With the aim of producing an enzyme optimized for d-tagatose production, three Bacillus stearothermophilus US100 l-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 °C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0–7.0 and an optimal activity around 50–65 °C, temperatures at which the enzyme was stable without addition of metal ions.     

Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH

The pH dependence of the Fe(III) reduction potential, E⁰′, for yeast cytochrome c peroxidase (yCcP) and three distal pocket mutants, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L), has been determined between pH 4 and 8. E⁰′ values at pH 7.0 for the yCcP, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L) are − 189, − 170, − 224, and − 146 mV, respectively. A heme-linked ionization in the reduced enzyme affects the reduction potential for yCcP and all three mutants. Apparent pK_A values for the heme-linked ionization are 7.5 ± 0.2, 6.5 ± 0.3, 6.4 ± 0.2, and 7.0 ± 0.3 for yCcP and the H52L, H52Q, and R48L/W51L/H52L mutants, respectively. A cooperative, two-proton ionization causing a spectroscopically-detectable transition was observed in the ferrous states of yCcP, CcP(H52L) and CcP(H52Q), with apparent pK_A values of 7.7 ± 0.2, 7.4 ± 0.1 and 7.8 ± 0.1, respectively. These data indicate that: (1) the distal histidine in CcP is not the site of proton binding upon reduction of the ferric CcP, (2) the distal histidine is not one of the two groups involved in the cooperative, two-proton ionization observed in ferrous CcP, and (3) the proton-binding site is not involved in the cooperative, two-proton ionization observed in the reduced enzyme.     

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80 °C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135 ± 6% and 193 ± 8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50 °C and about 45 °C respectively, while 65 °C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.     

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

Enzymatic hydrolysis of cellulose by the cellobiohydrolase domain of CelB from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus

The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-β-d-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80 °C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80 °C with half-lives of 23 h and 39 h, respectively, and retained >94% activity after 48 h at 70 °C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain.     

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

The Na⁺-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 (∼ 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated (∼ 68-90 kDa) with only 31-65% of WT l-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered (∼ 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at ∼ 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 °C or 37 °C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

Purification and biochemical characterization of Eumiliin from Euphorbia milii var. hislopii latex

A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatographic steps using DEAE-Sephacel and gel-filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS-PAGE under reducing conditions and gave one main peak at 29,814 KDa in MALDI-TOF/TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the Aα-chain of fibrinogen and, more slowly, the Bβ-chain. Its fibrinogenolytic activity is inhibited by β-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 °C; activity was completely lost at ?80 °C. Intraplantar injection of Eumiliin (1-25 μg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1-5 h after enzyme injection. Intraplantar injection of Eumiliin (1-25 μg/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection.     

Heat inactivation of Escherichia coli K12 MG1655: Effect of microbial metabolites and acids in spent medium

Aim

The effect of spent medium, obtained after different time-temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied.

Methods and results

Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time-temperature scenarios, i.e., (1) 30 °C to 55 °C at 0.14 °C/min, (2) 30 °C to 42 °C at 0.14 °C/min and (3) 30 °C to 42 °C at 0.8 °C/min. After the heat treatment, spent medium was filter-sterilized, non-stressed cells were added and inactivation experiments took place at 54 °C and 58 °C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium - compared to the unmodified BHI broth - are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance.

Conclusions

It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance.

Significance and impact of study

This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry.     

Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores

Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80 °C, and for the decolorization of indigo carmine at pH 8.0 and 60 °C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2 h at 37 °C. The apparent K_m of the enzyme displayed on spores was 443 ± 124 μM for ABTS with a V_max of 150 ± 16 U/mg spores. Notably, 1 mg of spores displaying B. subtilis laccase (3.4 × 10² U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2 h. The spore reactor (0.5 g of spores corresponding to 1.7 × 10⁵ U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60 °C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.     

Spectrophotometric quantification of horseradish peroxidase with o-phenylenediamine

The assay conditions for the spectrophotometric quantification of horseradish peroxidase (HRP) with the chromogenic substrate o-phenylenediamine (OPD) at 25 °C in the presence of hydrogen peroxide (H₂O₂) were optimized. With [OPD]₀ = 3.14 mM and [H₂O₂]₀ = 80 μM at pH 7.2, the initial formation of only one of the possible reaction products and intermediates, 2,3-diaminophenazine (DAP, λ_max = 417 nm), was observed. The rate of DAP formation during the first 30 min of reaction was followed spectrophotometrically and found to linearly depend on the HRP concentration between 5 and 45 pM under the conditions used.     

Role of K binding residues in stabilization of heme spin state of Leishmania major peroxidase

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.     

Human and rodent amyloid-β peptides differentially bind heme: Relevance to the human susceptibility to Alzheimer’s disease

Amyloid-β (Aβ) peptides are implicated in the neurodegeneration of Alzheimer’s disease (AD). We previously investigated the mechanism of neurotoxicity of Aβ and found that human Aβ (huAβ) binds and depletes heme, forming an Aβ-heme complex with peroxidase activity. Rodent Aβ (roAβ) is identical to huAβ, except for three amino acids within the proposed heme-binding motif (Site-H). We studied and compared heme-binding between roAβ and huAβ. Unlike roAβ, huAβ binds heme tightly (K_d = 140 ± 60 nM) and forms a peroxidase. The plot of bound (huAβ-heme) vs. unbound heme fits best to a two site binding hyperbola, suggesting huAβ possesses two heme-binding sites. Consistently, a second high affinity heme-binding site was identified in the lipophilic region (site-L) of huAβ (K_d = 210 ± 80nM). The plot of (roAβ-heme) vs. unbound heme, on the other hand, was different as it fits best to a sigmoidal binding curve, indicating different binding and lower affinity of roAβ for heme (K_d = 1 μM). The effect of heme-binding to site-H on heme-binding to site-L in roAβ and huAβ is discussed. While both roAβ and huAβ form aggregates equally, rodents lack AD-like neuropathology. High huAβ/heme ratio increases the peroxidase activity. These findings suggest that depletion of regulatory heme and formation of Aβ-heme peroxidase contribute to huAβ’s neurotoxicity in the early stages of AD. Phylogenic variations in the amino acid sequence of Aβ explain tight heme-binding to huAβ and likely contribute to the increased human susceptibility to AD.