Hybridization of modified-heme reconstitution and distal histidine mutation to functionalize sperm whale myoglobin

To modulate the physiological function of a hemoprotein, most approaches have been demonstrated by site-directed mutagenesis. Replacement of the native heme with an artificial prosthetic group is another way to modify a hemoprotein. However, an alternate method, mutation or heme reconstitution, does not always demonstrate sufficient improvement compared with the native heme enzyme. In the present study, to convert a simple oxygen storage hemoprotein, myoglobin, into an active peroxidase, we applied both methods at the same time. The native heme of myoglobin was replaced with a chemically modified heme 2 having two aromatic rings at the heme-propionate termini. The constructed myoglobins were examined for 2-methoxyphenol (guaiacol) oxidation in the presence of H2O2. Compared with native myoglobin, rMb(H64D.2) showed a 430-fold higher kcat/Km value, which is significantly higher than that of cytochrome c peroxidase and only 3-fold less than that of horseradish peroxidase. In addition, myoglobin-catalyzed degradation of bisphenol A was examined by HPLC analysis. The rMb(H64D.2) showed drastic acceleration (>35-fold) of bisphenol A degradation compared with the native myoglobin. In this system, a highly oxidized heme reactive species is smoothly generated and a substrate is effectively bound in the heme pocket, while native myoglobin only reversibly binds dioxygen. The present results indicate that the combination of a modified-heme reconstitution and an amino acid mutation should offer interesting perspectives toward developing a useful biomolecule catalyst from a hemoprotein.     

On-line chemiluminescence detection for isoelectric focusing of heme proteins on microchips

In this paper we present a sensitive chemiluminescence (CL) detection of heme proteins coupled with microchip IEF. The detection principle was based on the catalytic effects of the heme proteins on the CL reaction of luminol-H2O2 enhanced by para-iodophenol. The glass microchip and poly(dimethylsiloxane) (PDMS)/glass microchip for IEF were fabricated using micromachining technology in the laboratory. The modes of CL detection were investigated and two microchips (glass, PDMS/glass) were compared. Certain proteins, such as cytochrome c, myoglobin, and horseradish peroxidase, were focused by use of Pharmalyte pH 3-10 as ampholytes. Hydroxypropylmethylcellulose was added to the sample solution in order to easily reduce protein interactions with the channel wall as well as the EOF. The focused proteins were transported by salt mobilization to the CL detection window. Cytochrome c, myoglobin, and horseradish peroxidase were well separated within 10 min on a glass chip and the detection limits (S/N=3) were 1.2x10(-7), 1.6x10(-7), and 1.0x10(-10) M, respectively.     

Alkylamine-ligated H93G myoglobin cavity mutant: a model system for endogenous lysine and terminal amine ligation in heme proteins such as nitrite reductase and cytochrome f

His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, providing a remarkably versatile template for the preparation of model heme complexes. The investigation of model heme adducts is an important way to probe the relationship between coordination structure and catalytic function in heme enzymes. In this study, we have successfully generated and spectroscopically characterized the H93G Mb cavity mutant ligated with less common alkylamine ligands (models for Lys or the amine group of N-terminal amino acids) in numerous heme iron states. All complexes have been characterized by electronic absorption and magnetic circular dichroism spectroscopy in comparison with data for parallel imidazole-ligated H93G heme iron moieties. This is the first systematic spectral study of models for alkylamine- or terminal amine-ligated heme centers in proteins. High-spin mono- and low-spin bis-amine-ligated ferrous and ferric H93G Mb adducts have been prepared together with mixed-ligand ferric heme complexes with alkylamine trans to nitrite or imidazole as heme coordination models for cytochrome c nitrite reductase or cytochrome f, respectively. Six-coordinate ferrous H93G Mb derivatives with CO, NO, and O(2) trans to the alkylamine have also been successfully formed, the latter for the first time. Finally, a novel high-valent ferryl species has been generated. The data in this study represent the first thorough investigation of the spectroscopic properties of alkylamine-ligated heme iron systems as models for naturally occurring heme proteins ligated by Lys or terminal amines.     

Direct electrochemistry and electrocatalysis of heme proteins entrapped in agarose hydrogel films in room-temperature ionic liquids

The electrochemistry and electrocatalysis of a number of heme proteins entrapped in agarose hydrogel films in the room-temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) have been investigated. UV-vis and FTIR spectroscopy show that the heme proteins retain their native structure in agarose film. The uniform distribution of hemoglobin in agarose-dimethylformamide film was demonstrated by atomic force microscopy. Cyclic voltammetry shows that direct electron transfer between the heme proteins and glassy carbon electrode is quasi-reversible in [bmim][PF(6)]. The redox potentials for hemoglobin, myoglobin, horseradish peroxidase, cytochrome c, and catalase were found to be more negative than those in aqueous solution. The charge-transfer coefficient and the apparent electron-transfer rate constant for these heme proteins in [bmim][PF(6)] were calculated from the peak-to-peak separation as a function of scan rate. The heme proteins catalyze the electroreduction of trichloroacetic acid and tert-butyl hydroperoxide in [bmim][PF(6)]. The kinetic parameter I(max) (maximum current at saturation concentration of substrate) and the apparent K(m) (Michaelis-Menten constant) for the electrocatalytic reactions were evaluated.     

Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe(III)myoglobin, Fe(III)cytochrome c, Fe(III)cytochrome b5, and Fe(III)cytochrome b5 L47R

Unambiguous determination of metal atom oxidation state in an intact metalloprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic abundance mass distributions for one or more holoprotein charge states. The ion atom oxidation state is determined unequivocally as Fe(III) for each of four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobin, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution-phase oxidation state is conserved following electrospray to produce gas-phase ions). However, the same Fe(III) oxidation state in all four heme proteins is observed after prior reduction by sodium dithionite to produce Fe(II) heme proteins in solution: thus proving that oxygen was present during the electrospray process. Those results bear directly on the issue of similarity (or lack thereof) of solution-phase and gas-phase protein conformations. Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holoproteins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe(II)heme from the covalently bound heme in cytochrome c.     

Cytochrome c heme lyase can mature a fusion peptide composed of the amino-terminal residues of horse cytochrome c

It is shown that cytochrome c heme lyase (CCHL) attaches heme covalently to peptides composed of the N-terminal segment of cyt c fused to a non-heme containing protein, lending insight into the substrate specificity of CCHL and providing a new route to artificial heme proteins.     

Reactivities of mononuclear non-heme iron intermediates including evidence that iron(III)-hydroperoxo species is a sluggish oxidant

There is an intriguing, current controversy on the involvement of iron(III)-hydroperoxo species as a "second electrophilic oxidant" in oxygenation reactions by heme and non-heme iron enzymes and their model compounds. In the present work, we have performed reactivity studies of the iron-hydroperoxo species in nucleophilic and electrophilic reactions, with in situ-generated mononuclear non-heme iron(III)-hydroperoxo complexes that have been well characterized with various spectroscopic techniques. The intermediates did not show any reactivities in the nucleophilic (e.g., aldehyde deformylation) and electrophilic (e.g., oxidation of sulfide and olefin) reactions. These results demonstrate that non-heme iron(III)-hydroperoxo species are sluggish oxidants and that the oxidizing power of the intermediates cannot compete with that of high-valent iron(IV)-oxo complexes. We have also reported reactivities of mononuclear non-heme iron(III)-peroxo and iron(IV)-oxo complexes in the aldehyde deformylation and the oxidation of sulfides, respectively.     

Heme electron transfer in peroxidases: the propionate e-pathway

Computational modeling offers a new insight about the electron transfer pathway in heme peroxidases. Available crystal structures have revealed an intriguing arrangement of the heme propionate side chains in heme-heme and heme-substrate complexes. By means of mixed quantum mechanical/molecular mechanics calculations, we study the involvement of these propionate groups into the substrate oxidation in ascorbate peroxidase and into the heme to heme electron transfer in bacterial cytochrome c peroxidase. By selectively turning on/off different quantum regions, we obtain the electron transfer pathway which directly involves the porphyrin ring and the heme propionates. Furthermore, in ascorbate peroxidase the presence of the substrate appears to be crucial for the activation of the electron transfer channel. The results might represent a general motif for electron transfer from/to the heme group and change our view for the propionate side chains as simple electrostatic binding anchors. We name the new mechanism "the propionate e-pathway".     

Cytochrome c-crown ether complexes as supramolecular catalysts: cold-active synzymes for asymmetric sulfoxide oxidation in methanol

A series of supramolecular complexes of various cytochrome c proteins with 18-crown-6 derivatives behave as cold-active synzymes in the H2O2 oxidation of racemic sulfoxides. This interesting behavior contrasts with native functionality, where the employed proteins act as electron transfer carriers. ESI-MS. UV, CD, and Raman spectroscopic characterizations reveal that four or five 18-crown-6 molecules strongly bind to the surface of the cytochrome c and also that nonnatural low-spin hexacoordinate heme structures are induced in methanol. Significantly, crown ether complexation can convert catalytically inactive biological forms to catalytically active artificial forms. Horse heart, pigeon breast, and yeast cytochromes c all stereoselectively oxidize (S)-isomers of methyl tolyl sulfoxide and related sulfoxides upon crown ether complexation. These supramolecular catalysts show the highest efficiency and enantiomer selectivity at -40 degrees C in the H202-dependent sulfoxide oxidation, while oxidative decomposition of the heme moieties predominantly occurs at room temperature. The oxidation reactivity of the employed sulfoxides is apparently related to steric constraints and electrochemical oxidation potentials of their S=O bonds. Among the cytochrome c complexes, yeast cytochrome c demonstrates the lowest catalytic activity and degradation reactivity. It has a significantly different protein sequence, suggesting that crown ether complexation effectively activates heme coordination but may additionally alter the native backbone structure. The proper combination of cytochrome c proteins, 18-crown-6 receptors, and external circumstances can be used to successfully generate "protein-based supramolecular catalysts" exhibiting nonbiological reactivities.     

Two-state reactivity, electromerism, tautomerism, and "surprise" isomers in the formation of compound II of the enzyme horseradish peroxidase from the principal species, compound I

QM and QM/MM calculations on Compound II, the enigmatic species in the catalytic cycle of the horseradish peroxidase enzyme, reveal six low-lying isomers. The principal isomer is the triplet oxo-ferryl form (PorFe(IV)=O) that yields the hydroxo-ferryl isomer (PorFe(IV)-OH+). These are the only forms observed in experimental studies. Theory shows, however, that these are the least stable isomers of Compound II. The two most stable forms are the singlet and triplet states of the Por+*Fe(III)-OH electromer. In addition, theory reveals species never considered in heme enzymes: the singlet and triplet states of the Por+*Fe(III)-OH2 electromer. The computational results reproduce the experimental features of the known isomers and enable us to draw relationships and make predictions regarding the missing ones. For example, while the "surprise" species, singlet and triplet Por+*Fe(III)-OH2, have never been considered in heme chemistry, the calculated Fe-O bond lengths indicate that these isomers may have, in fact, been observed in one of the two opposing EXAFS studies reported previously. Furthermore, these ferric-aqua complexes could be responsible for the reported 18O exchange with bulk water. It is clear, therefore, that the role of Compound II in the HRP cycle is considerably more multi-faceted than has been revealed so far. Our suggested multi-state reactivity scheme provides a paradigm for Compound II species. The calculated M?ssbauer parameters may be helpful toward eventual characterization of these missing isomers of Compound II.     

On the formation, nature, stability and biological relevance of the primary reaction intermediates of myoglobins with hydrogen peroxide

The reaction between hydrogen peroxide and myoglobin (or haemoglobin) ferric haem is a two-electron redox process, yet the stable product is ferryl haem, retaining only one oxidizing equivalent. We have used SVD (singular value decomposition) and global spectroscopic analysis to examine the transient primary spectral intermediates in this reaction, which have been reported as either "compound 0"(ferric peroxide) or "compound I"(ferryl and porphyrin cation radical) types and which may precede the formation of ferrylmyoglobin. To test the hypothesis that the distal histidine facilitates ferryl formation we studied the myoglobin-like haemoglobin from the gastropod mollusc Aplysia fasciata, where this histidine is replaced by valine and its hydrogen bonding role is taken up by a non-homologous arginine. In this protein, consistent with the distal histidine hypothesis, a compound 0 intermediate is formed identified by an EPR spectrum typical of low spin ferric haem complexes. It is significantly more stable than any species seen with mammalian myoglobin. Thirdly, as ferryl haems and associated free radicals may play a role in disease, we have studied the action of myoglobin-peroxide mixtures towards external reductants. Even at a low pH, where ferrylmyoglobin is protonated and in its most reactive state, pre-incubation with reducing donors, including one-electron donors such as ferrocyanide, prior to peroxide addition renders both oxidizing equivalents available. The physiological antioxidant vitamin, ascorbate, is also able to trap both reactive species. Myoglobin can therefore act as a true ascorbate peroxidase. Ascorbate in vivo may be critical in controlling and preventing toxic side reactions of this and related haem proteins.     

Engineering tyrosine-based electron flow pathways in proteins: the case of aplysia myoglobin

Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed.     

Generation of High-Oxidation States of Myoglobin in the Nanosecond Time-Scale by Laser Photoionization

Abstract— The 248 nm laser flash photolysis of myoglobin in various redox states (oxy, met and ferryl) in neutral aqueous solution yielded hydrated electrons with concurrent changes in the visible absorption spectrum of the heme. The results could be ascribed to the photoionization of both the peptide and the heme group, in approximately equal yields. The ionization of met- and ferrylmyoglobin was biphotonic, but that of oxymyoglobin was a mixture of mono- and biphotonic processes. Using appropriate electron and radical scavengers, the changes in the heme absorption could be investigated at times ≥100 ns and were shown to be associated with a +1 increase of the formal oxidation state of the heme. Using this method, the formal iron(V) state of native myoglobin could be spectroscopically characterized for the first time. Its absorption, blue-shifted and less intense relative to the ferryl state, is reminiscent of that of the compound I of peroxidases, which contains a ferryl-oxo (iron[IV]) group and a porphyrin radical cation. On this basis, the same structure is proposed for the formal iron(V) state of native myoglobin. The transition from oxy- to metmyoglo-bin took -5 μs, which may reflect the kinetics of exchange of oxygen with water as ligand. The transitions from the met to the ferryl state, and from ferryl to iron(V) states were faster (∽250 ns), consistent with processes that involve proton or electron movements but no changes in the iron coordination state.     

Spectroscopic and quantum chemical studies on low-spin FeIV=O complexes: Fe-O bonding and its contributions to reactivity

High-valent FeIV=O species are key intermediates in the catalytic cycles of many mononuclear non-heme iron enzymes and have been structurally defined in model systems. Variable-temperature magnetic circular dichroism (VT-MCD) spectroscopy has been used to evaluate the electronic structures and in particular the Fe-O bonds of three FeIV=O (S = 1) model complexes, [FeIV(O)(TMC)(NCMe)]2+, [FeIV(O)(TMC)(OC(O)CF3)]+, and [FeIV(O)(N4Py)]2+. These complexes are characterized by their strong and covalent Fe-O pi-bonds. The MCD spectra show a vibronic progression in the nonbonding --> pi* excited state, providing the Fe-O stretching frequency and the Fe-O bond length in this excited state and quantifying the pi-contribution to the total Fe-O bond. Correlation of these experimental data to reactivity shows that the [FeIV(O)(N4Py)]2+ complex, with the highest reactivity toward hydrogen-atom abstraction among the three, has the strongest Fe-O pi-bond. Density functional calculations were correlated to the data and support the experimental analysis. The strength and covalency of the Fe-O pi-bond result in high oxygen character in the important frontier molecular orbitals (FMOs) for this reaction, the unoccupied beta-spin d(xz/yz) orbitals, that activates these for electrophilic attack. An extension to biologically relevant FeIV=O (S = 2) enzyme intermediates shows that these can perform electrophilic attack reactions along the same mechanistic pathway (pi-FMO pathway) with similar reactivity but also have an additional reaction channel involving the unoccupied alpha-spin d(z2) orbital (sigma-FMO pathway). These studies experimentally probe the FMOs involved in the reactivity of FeIV=O (S = 1) model complexes resulting in a detailed understanding of the Fe-O bond and its contributions to reactivity.     

Peroxidase activity of enzymes bound to the ends of single-wall carbon nanotube forest electrodes

This communication reports the first example, to our knowledge, of enzymes covalently attached onto the ends of vertically oriented single-wall carbon nanotube (SWNT) forest arrays used as electrodes. Quasi-reversible Fe^III/Fe^II voltammetry was observed for the iron heme enzymes myoglobin and horseradish peroxidase coupled to carboxylated ends of the nanotube forests by amide linkages. Results suggest that the “trees” in the nanotube forest behaved electrically similar to a metal, conducting electrons from the external circuit to the redox sites of the enzymes. Electrochemically manifested peroxidase activity of myoglobin and horseradish peroxidase attached to the SWNT forests was demonstrated, with detection limits for hydrogen peroxide in buffer solutions of ∼100 nM. These prototype SWNT-forest biosensors are easy to prepare, and enzyme layers were stable for weeks.     

Heme CD as a probe for monitoring local structural changes in hemeproteins: Alkaline transition in hemeproteins

Structural change due to acid-alkaline transition in hemeproteins were monitored by circular dichroism measurements in the Soret region. It was observed that in cytochrome c and horseradish peroxidase, alkaline transition results in a large change in the heme CD due to significant conformational change in the heme cavity region. In metmyoglobin a simple protolytic mechanism associated with alkaline transition involves very small conformational changes.     

Reaction Mechanism of Enzyme Studied by Pulse Radiolysis

By virtue of its ability to generate hydrated electrons (e_aq ^?) and various radicals as reductants, the pulse radiolysis technique has been employed for investigating the mechanism of action of peroxidase, cytochrome P-450, and cytochrome oxidase. The oxy forms of hemoproteins, such as myoglobin, peroxidase, and cytochrome P-450, were reduced by hydrated electrons to form the higher oxidation states of these hemoproteins. From these results, the reactive oxygen intermediate of cytochrome chrome P-450 is discussed. The reduction of cytochrome oxidase by the 1-methylnicotinamide radical was investigated. A decrease of the 830-nm band was detected due to the reduction of “visible” copper. After the first phase of the reduction of copper, the return of the 830-nm band corresponding to oxidation of copper was observed. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme α increased. This suggests that 1-methylnicotinamide radical reacts with the “visible” copper and subsequently flows to heme α by intramolecular migration.     

Precise Design of Artificial Cofactors for Enhancing Peroxidase Activity of Myoglobin: Myoglobin Mutant H64D Reconstituted with a “Single‐Winged Cofactor” Is Equivalent to Native Horseradish Peroxidase in Oxidation Activity

H64D myoglobin mutant was reconstituted with two different types of synthetic hemes that have aromatic rings and a carboxylate‐based cluster attached to the terminus of one or both of the heme‐propionate moieties, thereby forming a “single‐winged cofactor” and “double‐winged cofactor,” respectively. The reconstituted mutant myoglobins have smaller K_m values with respect to 2‐methoxyphenol oxidation activity relative to the parent mutant with native heme. This suggests that the attached moiety functions as a substrate‐binding domain. However, the k_cat value of the mutant myoglobin with the double‐winged cofactor is much lower than that of the mutant with the native heme. In contrast, the mutant reconstituted with the single‐winged cofactor has a larger k_cat value, thereby resulting in overall catalytic activity that is essentially equivalent to that of the native horseradish peroxidase. Enhanced peroxygenase activity was also observed for the mutant myoglobin with the single‐winged cofactor, thus indicating that introduction of an artificial substrate‐binding domain at only one of the heme propionates in the H64D mutant is the optimal engineering strategy for improving the peroxidase activity of myoglobin.     

Studies on heme binding in myoglobin,hemoglobin, and cytochrome c by ion spray mass spectrometry

The ion spray mass spectra of three representative heme-containing proteins were studied, with an emphasis on results obtained under neutral (pH 7) aqueous conditions. The noncovalently bound heme in myoglobin and hemoglobin may be readily distinguished from the covalently bound heme prosthetic group attached to cytochrome c by using collisioninduced dissociation in the free-jet expansion region of the mass spectrometer as well as in the collision quadrupole with premass selection. The charge state of iron in the expelled heme from myoglobin and hemoglobin appears to be 3+ but 2f for heme expelled from cytochrome c.     

DEMONSTRATION THAT PHOSPHORESCENT 6-BROMO-2-NAPHTHYL SULFATE CAN BE USED TO PROBE HEME ACCESSIBILITY IN HEME PROTEINS

The phosphorescence properties of 6-bromo-2-naphthyl sulfate (BNS) in aqueous solution were studied. The phosphorescence lifetime is several hundred microseconds and is self-quenched. Although a fluorescent photoproduct is formed from BNS, it does not interfere with the decay properties of triplet-state BNS and its utility as a probe of the accessibility of the heme group in heme proteins. Quenching of BNS phosphorescence does not occur for the non-heme protein lysozyme and apomyoglobin but occurs by a dynamic mechanism with a quenching constant of 1-2 x 10(9) M-1 s-1 for cytochrome c and myoglobin and with a quenching constant of 6.2 x 10(9) M-1 s-1 for protoporphyrin IX. The phosphorescence of an inclusion complex of 1-bromonaphthalene and beta-cyclodextrin is not quenched by heme-containing proteins. The temperature and viscosity dependencies of the rate with which BNS phosphorescence is quenched by microperoxidase-11 are consistent with unit quenching efficiency. These results indicate that quenching of BNS phosphorescence occurs only upon contact with the quencher, and the quenching constant can be used to assess the degree of accessibility of the heme group.