Microperoxidase-11 Research Articles

Formation of Iron(II)–Nitrosoalkane Complexes: A New Activity of Microperoxidase 8

Microperoxidase 8 (MP8) is a heme octapeptide, obtained by enzymatic hydrolysis of heart cytochrome c, in which a histidine is axially coordinated to the heme iron, and acts as its fifth ligand. It exhibits two kinds of activities: a peroxidase-like activity and a cytochrome P450-like activity. We here show that MP8 is not only able to oxidize various aliphatic and aromatic hydroxylamines with the formation of MP8-Fe(II)-nitrosoalkane or -arene complexes absorbing around 414 nm, but also that these complexes can be obtained by reduction of nitroalkanes. This is the first example of fully characterized iron(II)-metabolite complexes of MP8. Such complexes constitute good models for those obtained upon oxidation of amphetamine or macrolids by cytochromes P450. In addition, this is a new catalytic activity of MP8, which validates the use of this mini-enzyme as a convenient model for hemoproteins of interest in toxicology and pharmacology such as cytochromes P450 and peroxidases.

Heme-(hydro)peroxide mediated O- and N-dealkylation. A study with microperoxidase.

The mechanism of microperoxidase-8 (MP-8) mediated O- and N-dealkylation was investigated. In the absence of ascorbate (peroxidase mode), many unidentified polymeric products are formed and the extent of substrate degradation correlates (r = 0.94) with the calculated substrate ionization potential, reflecting the formation of radical intermediates. In the presence of ascorbate (P450 mode) formation of polymeric products is largely prevented but, surprisingly, dealkylation is not affected. In addition, aromatic hydroxylation and oxidative dehalogenation is observed. The results exclude a radical mechanism and indicate the involvement of a (hydro)peroxo-iron heme intermediate in P450-type of heteroatom dealkylation.

Investigation of the Mechanism of Action of Microperoxidase-11, (MP11), a Potential Anti-cataract Agent, with Hydrogen Peroxide and Ascorbate

The interaction of hydrogen peroxide, ascorbate and microperoxidase-11 (MP11), a ferriheme undecapeptide derived from cytochrome c, has been investigated using spectrophotometry, oxymetry, electron paramagnetic resonance (EPR), and mass spectroscopy techniques. It is shown that in 50m M phosphate pH 7.0–7.4 in the absence of other reactants H2O2induces a concentration-dependent decrease in absorption at the Soret band (399nm) of the microperoxidase, with concomitant H2O2decomposition and oxygen evolution. The reaction causes irreversible heme degradation, concomitant with loss of enzymatic activity. Ascorbate effectively protects MP11 from degradation and inhibits oxygen evolution. At ascorbate concentrations greater than that of H2O2, microperoxidase degradation is almost completely prevented. Mass spectrometry showed that H2O2oxidizes the microperoxidase to a monooxygenated product, which did not form if ascorbate was included in the reaction system. There appears to be a 1:1 relationship between H2O2degradation and ascorbate oxidation. EPR experiments revealed that an ascorbate radical was formed during the reaction. These reactions may be described by a scheme where a putative ‘compound I’ of the microperoxidase is reduced by ascorbate back to the original redox state (ferric) of the peroxidase in two one-electron steps, concomitantly with oxidation of the ascorbate to an ascorbate radical or in one two-electron transfer step forming dehydroascorbate. In the absence of ascorbate, the ‘compound I’ reacts further with the peroxide causing microperoxidase degradation and partial oxygen evolution. These observations are relevant to the interaction of ferrihemes with H2O2and ascorbic acid and may be pertinent for the potential application of MP11 as an anti-cataract agent.

UV resonance Raman probe of heme‐bound imidazole established by 15N‐labeling of hemoglobin

AbstractRecent studies of Cu, Zn superoxide dismutase, and of zinc‐finger peptides have established that histidine ligands can be detected in ultraviolet resonance Raman (UVRR) spectra, following NH/D exchange of the imidazole. UVRR spectroscopy therefore offers promise for monitoring histidine ligation in heme proteins. In this work, we characterize heme‐bound histidine UVRR bands for N‐acetyl‐microperoxidase‐8 (MP‐8) and microperoxidase‐11 (MP‐11), and also for hemoglobin (Hb). The Hb UVRR spectra are dominated by tyrosine and tryptophan contributions, but a band appears at 1340 cm−1 in D2O solution, which is assigned to a mode of Fe‐bound imidazole. This band shifted 24 cm−1 in protein which was labeled with 15N via expression of the Hb gene in E. coli grown on 15NH4+. In MP‐11, the position of this band is insensitive to ligation or oxidation state changes, but it is 2 cm−1 lower in deoxyHb than in the CO adduct. This shift may reflect mechanical forces on the proximal histidine in the T state, and/or changes in its H‐bonding.

A Mechanism for Oxygen Exchange between Ligated Oxometalloporphinates and Bulk Water

Oxygen exchange between high-valent metal–oxo complexes and bulk water has been monitored for nonligated model porphyrins (hemin, FeTDCPPS, MnTMPyP) and the axially ligated microperoxidase-8 (MP-8). Exchange extents up to 90% were measured for MP-8 in spite of the presence of an axial histidine ligand and accompanied by the formation of nonlabelled H2O2 from H218O2. These results point to the existence of a mechanism for oxygen exchange between the high-valent iron–oxo complex and the solvent different from the so-called “oxo-hydroxo tautomerism.” Regeneration of the primary oxidant, H2O2, and oxygen exchange by axially ligated porphyrins can be explained by a mechanism involving the reversibility of compound I formation.

3D structure of microperoxidase-11 by NMR and molecular dynamic studies

Microperoxidase-11 (MP11) is an undecapeptide, obtained from enzymatic cleavage of cytochrome c, which still exhibits peroxidase activity. It retains residues 11–21 of the protein, the heme-c group and His18 as fifth ligand at the iron atom. We studied MP11 in the paramagnetic, Fe(III) low-spin state by NMR and constrained molecular dynamics (MD). Well-resolved 1H and 13C spectra were obtained in water–trifluoroethanol and water–methanol mixtures with different axial ligands to the iron, i.e. imidazole, NH3, OH, CN, at different temperatures from −15 to +25 °C. All the protons of the heme and the peptide moiety were assigned by using TOCSY and NOESY experiments. Some stereospecific assignments were also performed and eight coupling constants between the amide and the α-protons were measured. Seventy-one inter-residue and heme peptide and 40 intra-residue NOE interactions were translated into inter-proton distances. Ten structures satisfying these distance constraints to within ±0.4 Å were obtained and showed that almost the whole peptide (segment 12–19) can form a right-handed helix. The envelope structure of the peptide around the heme is well defined based on a number of NOE interactions between heme and peptide protons. Variable-temperature experiments were performed in order to obtain information on the protons affected by the paramagnetic shift. An inverted temperature dependence (Curie law) was observed for the heme and meso protons, except for 3-Me, when the sixth axial ligand is NH3 or imidazole. The pattern of 13C hyperfine shifts of heme methyl groups, similar in MP11 and cytochrome c, suggests that the orientation of His ring along the α–γ meso axis is conserved in MP11, which is consistent with MD calculations. Copyright © 2000 John Wiley & Sons, Ltd.

Covalently modified microperoxidases as heme-peptide models for peroxidases

Microperoxidase-8 (MP8) and microperoxidase-9 (MP9) have been covalently modified by attachment of proline-containing residues to the amino terminal peptide chain in order to obtain new peroxidase model systems. The catalytic activities of these derivatives in the oxidation of p-cresol by hydrogen peroxide have been compared to that of MP8. The presence of steric hindrance above the heme reduces the formation rate of the catalytically active species, while the reactivity is increased when the amino group of a proline residue is close to the iron. The modification of the catalyst affects the rate of degradation processes undergone by the heme group during catalysis. A bulky aromatic group on the distal side decreases the stability of the complex because it reduces the mobility of a phenoxy radical species formed during catalysis, while the presence of proline residues increases the number of turnovers of the heme catalysts before degradation. The complex Pro 2-MP8 obtained by addition of two proline residues to MP8 exhibits the best catalytic performance in terms of activity and chemical stability.

Controlled electrocatalysis by microperoxidase-11 and Au-nanoparticle superstructures on conductive supports

Abstract Three-dimensional superstructures, multilayer-arrays consisting of Au-nanoparticles (13±1 nm) and crosslinked by microperoxidase-11 (MP-11), were assembled on transparent ITO conductive glass supports. The structures of the assemblies were confirmed by spectroscopic and electrochemical analyses, which revealed that each Au-particle is associated with ca. 50 MP-11 molecules. The multilayered MP-11/Au-particle systems act as electrocatalysts for the reduction of H2O2. The number of MP-11 ∣ Au-particle layers associated with the electrode controls the resulting electrocatalytic currents. The controllable number of MP-11 ∣ Au-particle layers associated with the electrode enables the control of the effectiveness of the electrocatalytic process and tuning of the sensitivity of the H2O2-sensing interface.

Conversion of pentahalogenated phenols by microperoxidase-8/H2O2 to benzoquinone-type products.

This study reports the microperoxidase-8 (MP8)/H2O2-catalyzed dehalogenation of pentafluorophenol and pentachlorophenol, compounds whose toxic effects and persistence in the environment are well documented. The primary products of this dehalogenation reaction appear to be the corresponding tetrahalo-p-benzoquinones. Under the conditions used, the fluorinated phenol and its intermediate products are more susceptible to degradation than the corresponding chlorinated analogue and its products. The main degradation products of tetrachloro-p-benzoquinone and tetrafluoro-p-benzoquinone were identified as trichlorohydroxy-p-benzoquinone and trifluorohydroxy-p-benzoquinone, respectively. This secondary conversion of tetrafluoro-p-benzoquinone and tetrachloro-p-benzoquinone was not mediated by MP8, but was driven by H2O2. Evidence is presented for a mechanism where H2O2 molecules and not hydroxide anions are the reactive nucleophilic species attacking the tetrahalo-p-benzoquinones. In addition to the formation of the trihalohydroxy-p-benzoquinones, the formation of adducts of the tetrahalo-p-benzoquinone products with ethanol, present in the incubation medium, was observed. The adduct from the reaction of tetrachloro-p-benzoquinone with ethanol was isolated and identified as trichloroethoxyquinone. Thus, the present paper describes a system in which the formation of tetrahalo-p-benzoquinone-type products by an oxidative heme-based catalyst could be unequivocally demonstrated.

Fully integrated biocatalytic electrodes based on bioaffinity interactions

Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD +-dyad for the binding of the NAD +-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD +-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone–NAD + monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N 6-(2-aminoethyl)-NAD + to the monolayer. The interface modified with the PQQ-NAD +-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or Co II-protoporphyrin IX reconstituted myoglobin (Co II–Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and Co II–Mb show catalytic activities for NO 3 − reduction and acetylenedicarboxylic acid hydrogenation, respectively. Two Fe III-protoporphyrin IX units are reconstituted into a four α-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the Fe III-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.

Crosslinked integrated enzyme electrodes for bioelectronic applications

Affinity interactions between electron-relay-functionalized electrodes and redox enzymes form stable protein-monolayer assemblies that upon crosslinking yield integrated, electrically contacted, enzyme electrodes. A functionalized monolayer consisting of pyrroloquinoline quinone, covalently linked to N6-(2-aminoethyl)-NAD+ (PQQ-NAD monolayer), was assembled on a Au electrode. The NAD - cofactor units form an affinity complex with the NAD - dependent enzyme lactate dehydrogenase (LDH). Crosslinking of the affinity complex with glutaric dialdehyde yields a stable crosslinked two-dimensional array of the enzyme on the electrode surface. The resulting PQQ-NAD / LDH crosslinked protein array is electrically contacted and reveals bioelectrocatalytic features. The electrocatalyzed oxidation of biocatalytically generated NADH by the PQQ components yields an amperometric response whose magnitude is related to the concentration of lactate. Similarly. an integrated, electrically contacted, enzyme electrode was generated by the crosslinking of a microperoxidase-11 (MP-11) and nitrate reductase (NR) affinity complex on a Au electrode with glutaric dialdehyde. Bioelectrocatalyzed reduction of nitrate (NOT) to nitrite (NO 2 - ) by the crosslinked enzyme electrode proceeds by the MP-11-mediated reduction of NR. The enzyme electrode stimulates the electrocatalyzed reduction of NO 3 - with a current yield corresponding to ca. 85%.

Crosslinked integrated enzyme electrodes for bioelectronic applications

Affinity interactions between electron-relay-functionalized electrodes and redox enzymes form stable protein-monolayer assemblies that upon crosslinking yield integrated, electrically contacted, enzyme electrodes. A functionalized monolayer consisting of pyrroloquinoline quinone, covalently linked to N6-(2-aminoethyl)-NAD* (PQQ-NAD* monolayer), was assembled on a Au electrode. The NAD*-cofactor units form an affinity complex with the NAD*-dependent enzyme lactate dehydrogenase (LDH). Crosslinking of the affinity complex with glutaric dialdehyde yields a stable crosslinked two-dimensional array of the enzyme on the electrode surface. The resulting PQQ-NAD*/LDH crosslinked protein array is electrically contacted and reveals bioelectrocatalytic features. The electrocatalyzed oxidation of biocatalytically generated NADH by the PQQ components yields an amperometric response whose magnitude is related to the concentration of lactate. Similarly, an integrated, electrically contacted, enzyme electrode was generated by the crosslinking of a microperoxidase-11 (MP-11) and nitrate reductase (NR) affinity complex on a Au electrode with glutaric dialdehyde. Bioelectrocatalyzed reduction of nitrate (NO3¯) to nitrite (NO2¯) by the crosslinked enzyme electrode proceeds by the MP-11-mediated reduction of NR. The enzyme electrode stimulates the electrocatalyzed reduction of NO3¯ with a current yield corresponding to ca. 85%.

Membrane covered carbon paste electrode for the electrochemical determination of peroxidase and microperoxidase in a flow system

Electrocatalytical reduction of hydrogen peroxide in a flow system was performed by horseradish peroxidase (HRP) and microperoxidase-11 (MP-11) on membrane covered modified carbon paste electrodes (MCPE). MCPE contained the mediator 1-( N, N-dimethylamine)-4-(4-morpholine)benzene (AMB) dissolved in paraffin. The electrocatalytical process proceeded at 0.0 V vs. 0.1 M NaCl Ag/AgCl. Half-time of the electrocatalytic current in flow system was dependent on enzyme and electrode type. In the case of HRP it was 2.1 and 8.5 min, respectively, for bare and membrane covered and with 1.7% of mediator modified electrode. Decreasing the AMB amount 10 times changed the half-time only a little. The kinetics of MP-11 current evolution was not limited by MP-11 transport through the membrane since the half-time change was small for bare and membrane covered electrode. The dependence of electrocatalytical current on hydrogen peroxide showed hyperbolic form at different AMB amount. The largest current for HRP and MP-11 was measured at carbon paste loading by 1.7% of mediator. Half-concentration of hydrogen peroxide changed from 0.04–0.12 mM and from 0.18 to 1.4 mM, respectively, for HRP and MP-11. In the case of HRP the largest ratio between membrane free and membrane covered MCPE current was at 0.88% of mediator loading. A leakage of AMB from membrane free MCPE was fast in the first 10 min and later stabilized. The total amount of AMB liberated from the electrode in the period of 2.5 h corresponded to 12% of the total mediator quantity in MCPE. The electrocatalytical current decreased only 16% over this period of time. The electrode covering by membrane suppressed the fast phase of mediator leakage, the total amount of liberated mediator was similar to the bare electrode, but the electrode current decreased only 5%.

Manganese Microperoxidase-8

Demetalation of Fe(III) microperoxidase-8 (MP8) by anhydrous HF gives metal-free MP8, a convenient starting material for a wide variety of metal-substituted MP8 derivatives, including Mn(III)MP8. Mn(III)MP8 was produced by treatment of metal-free MP8 with manganous acetate in aerated aqueous solution; it was characterized by mass spectrometry and UV−visible absorption spectroscopy. Resonance Raman spectra suggest that Mn(III)MP8 contains histidine and water axial ligands at neutral pH. The Mn(IV)=O derivative is readily prepared by oxidation of Mn(III)MP8 with hydrogen peroxide or Ru(bpy)_3^(3+) (bpy = 2,2‘-bipyridine).

A biofuel cell based on pyrroloquinoline quinone and microperoxidase-11 monolayer-functionalized electrodes

A pyrroloquinoline quinone (PQQ) monolayer-functionalized-Au-electrode and a microperoxidase-11 (MP-11)-modified Au-electrode are used as catalytic anode and cathode in a biofuel cell element, respectively. The cathodic oxidizer is H 2O 2 whereas the anodic fuel-substrate is 1,4-dihydronicotinamide adenine dinucleotide, NADH. The PQQ-monolayer electrode catalyzes the oxidation of NADH in the presence of Ca 2+ ions. The MP-11-functionalized electrode catalyzes the reduction of H 2O 2. The biofuel cell generates an open-circuit voltage, V oc, of ca. 320 mV and a short-circuit current density, I sc, of ca. 30 μA·cm −2. The maximum electrical power, W max, extracted from the cell is 8 μW at an external load of 3 kΩ. The fill factor of the biofuel cell, f= W max· I sc −1· V oc −1, is ca. 25%.

Synthesis and Physical Characterization of Novel Heme-Based Model Systems for Photoinitiated Electron Transfer. 1. Complexation of a RuProHis Bifunctional Peptide and Microperoxidase-11

This paper describes the synthesis and initial physical characterization of a new type of model system for the investigation of photoinitiated electron transfer (ET) in proteins and polypeptides. This system is based upon a derivative of a heme-containing digestion product of cytochrome c, microperoxidase-11 (MP-11). The vacant axial position of the five-coordinate heme of MP-11 is coordinated to the terminal histidine of a dipeptide having a photoactive ruthenium tris(bipyridine) moiety at its other end (RuProHis). Photoexcitation of the ruthenium group results in rapid (kET > 10 7 s -1 ), reversible ET to the ferric heme. The equilibrium properties of MP-11 and the MP-11/Ru(peptide) complex were characterized with optical absorption, luminescence, and resonance Raman (RR) spectroscopies. Molecular modeling was also employed to examine the structure and energetics of the equilibrium species. Clear evidence for reversible photoinduced ET was observed in time-resolved luminescence and transient resonance Raman studies of the MP-11/RuProHis samples.

Synthesis and Physical Characterization of Novel Heme-Based Model Systems for Photoinitiated Electron Transfer. 2. Direct Ruthenation of Microperoxidase-11

This paper describes the design, synthesis, and initial characterization of a second type of model electron transfer system based upon microperoxidase-11 (MP-11) that differs significantly from those described in the preceding article. In this system, a photoactive ruthenium tris(bipyridyl) donor is covalently attached directly to MP-11. Peptide digestion followed by chromatographic analysis was used to establish that the ruthenium moiety bonds to the lysine residue of the MP-11 peptide. This arrangement eliminates complications arising from the equilibrium complexation of ruthenium donor and heme acceptor required for the model systems described in the previous article and leaves an axial position in the octahedral geometry of the heme iron open for ligation. Various optical spectroscopies and molecular modeling calculations were employed to characterize the equilibrium properties and interchromophore interactions of RuMP-11. Preliminary luminescence-quenching, transient absorption, and transient resonance Raman studies demonstrated that rapid reversible electron transfer can be photoinitiated in this system.

Microperoxidase-11-mediated reduction of hemoproteins: electrocatalyzed reduction of cytochrome c, myoglobin and hemoglobin and electrocatalytic reduction of nitrate in the presence of cytochrome-dependent nitrate reductase

Microperoxidase-11 (MP-11) assembled onto an Au electrode catalyzes the electroreduction of a series of hemoproteins: cytochrome c (Cyt. c) myoglobin (Mb) and hemoglobin (Hb). The electrical contact between MP-11 and the hemoproteins originates from intermolecular complexes between MP-11 and the different hemoproteins. The MP-11-functionalized electrode catalyzes the reduction of cytochrome-dependent nitrate reductase and mediates the bioelectrocatalyzed reduction of nitrate to nitrite. The systems provide assemblies mimicking inter-protein electron transfer in hemoproteins.

Microperoxidase/H2O2-mediated alkoxylating dehalogenation of halophenol derivatives in alcoholic media.

The results of this study report the H2O2-driven microperoxidase-8 (MP8)-catalyzed dehalogenation of halophenols such as 4-fluorophenol, 4-chlorophenol, 4-bromophenol, and 2-fluorophenol in alcoholic solvents. In methanol, the conversion of the para-halophenols and 2-fluorophenol to, respectively, 4-methoxyphenol and 2-methoxyphenol, as the major dehalogenated products is observed. In ethanol, 4-ethoxyphenol is the principal dehalogenated product formed from 4-fluorophenol. Two mechanisms are suggested for this MP8-dependent alkoxylating dehalogenation reaction. In one of these mechanisms the oxene resonant form of compound I of MP8 is suggested to react with methanol forming a cofactor-peroxide-alkyl intermediate. This intermediate reacts with the reactive pi-electrons of the substrate, leading to the formation of the alkoxyphenols and the release of the fluorine substituent as fluoride anion.

Direct electrochemistry of microperoxidase-11 at gold electrodes modified by self-assembled monolayers of 4,4′-dithiodipyridine and 1-octadecanethiol

Abstract Direct redox conversion of microperoxidase-11 (MP-11) was demonstrated and studied by cyclic voltammetry on a gold electrode modified by a self-assembled monolayer (SAM) of 4,4′-dithiodipyridine (DTDP) or 1-octadecanethiol (ODT). The formation of the DTDP- and ODT-based SAMs on gold was evidenced by ellipsometry. The formal heterogeneous electron transfer rate constant of MP-11 redox conversion on the DTDP-modified electrode was 2.0(±0.3) × 10 −3 cm s −1 . The sigmoidal shape of the MP-11 voltammograms on the partly ODT-coated Au surface suggested the electron transfer at the monolayer-based microelectrode assemblies. Distinct mechanisms of the MP-11 redox conversion on the DTDP- and ODT-modified Au electrodes were discussed.