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.     

Unusual ligand discrimination by a myoglobin reconstituted with a hydrophobic domain-linked heme

New, reconstituted horse heart myoglobins possessing a hydrophobic domain at the terminal of the two heme propionate side chains were constructed. The O2 and CO bindings for the reconstituted deoxymyoglobins were examined in detail by laser flash photolysis and stopped-flow rapid mixing techniques. The artificially created domain worked as a barrier against exogenous ligand penetration into the heme pocket, whereas the bound O2 was stabilized in the reconstituted myoglobin as well as in the native one. In contrast, the CO dissociation rate for the reconstituted myoglobin increased by 20-fold compared to the native protein, suggesting that the incorporation of the hydrophobic domain onto the heme pocket perturbs the distal-site structure of the reconstituted myoglobin. As a result, the substantial ligand selectivity for the reconstituted myoglobin significantly increases in favor of O2 over CO with the M' value (= KCO/KO2) of 0.88, whereas, to the best of our knowledge, there is no myoglobin mutant in which the O2 affinity exceeds the CO one. The present work concludes that the O2 selectivity of myoglobin over CO is markedly improved by chemically modifying the heme propionates without any mutation of the amino acid residues in the distal site.     

Crystal structure and peroxidase activity of myoglobin reconstituted with iron porphycene

The incorporation of an artificially created metal complex into an apomyoglobin is one of the attractive methods in a series of hemoprotein modifications. Single crystals of sperm whale myoglobin reconstituted with 13,16-dicarboxyethyl-2,7-diethyl-3,6,12,17-tetramethylporphycenatoiron(III) were obtained in the imidazole buffer, and the 3D structure with a 2.25-A resolution indicates that the iron porphycene, a structural isomer of hemin, is located in the normal position of the heme pocket. Furthermore, it was found that the reconstituted myoglobin catalyzed the H2O2-dependent oxidations of substrates such as guaiacol, thioanisole, and styrene. At pH 7.0 and 20 degrees C, the initial rate of the guaiacol oxidation is 11-fold faster than that observed for the native myoglobin. Moreover, the stopped-flow analysis of the reaction of the reconstituted protein with H2O2 suggested the formation of two reaction intermediates, compounds II- and III-like species, in the absence of a substrate. It is a rare example that compound III is formed via compound II in myoglobin chemistry. The enhancement of the peroxidase activity and the formation of the stable compound III in myoglobin with iron porphycene mainly arise from the strong coordination of the Fe-His93 bond.     

Ligand binding properties of myoglobin reconstituted with iron porphycene: unusual O2 binding selectivity against CO binding

Sperm whale myoglobin, an oxygen storage hemoprotein, was successfully reconstituted with the iron porphycene having two propionates, 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)porphycenatoiron. The physicochemical properties and ligand bindings of the reconstituted myoglobin were investigated. The ferric reconstituted myoglobin shows the remarkable stability against acid denaturation and only a low-spin characteristic in its EPR spectrum. The Fe(III)/Fe(II) redox potential (-190 mV vs NHE) determined by the spectroelectrochemical measurements was much lower than that of the wild-type. These results can be attributed to the strong coordination of His93 to the porphycene iron, which is induced by the nature of the porphycene ring symmetry. The O2 affinity of the ferrous reconstituted myoglobin is 2600-fold higher than that of the wild-type, mainly due to the decrease in the O2 dissociation rate, whereas the CO affinity is not so significantly enhanced. As a result, the O2 affinity of the reconstituted myoglobin exceeds its CO affinity (M' = K(CO)/K(O2) < 1). The ligand binding studies on H64A mutants support the fact that the slow O2 dissociation of the reconstituted myoglobin is primarily caused by the stabilization of the Fe-O2 sigma-bonding. The IR spectra for the carbon monoxide (CO) complex of the reconstituted myoglobin suggest several structural and/or electrostatic conformations of the Fe-C-O bond, but this is not directly correlated with the CO dissociation rate. The high O2 affinity and the unique characteristics of the myoglobin with the iron porphycene indicate that reconstitution with a synthesized heme is a useful method not only to understand the physiological function of myoglobin but also to create a tailor-made function on the protein.     

Electrochemistry of monoazahemin reconstituted myoglobin at an indium oxide electrode

Monoazahemin reconstituted myoglobin was prepared and its electrochemical behavior was studied in comparison with native myoglobin. For both myoglobins well-defined voltammograms were clearly obtained at highly hydrophilic surfaces of indium oxide electrodes. Although monoazahemin showed a more positive redox potential than hemin (measured in methanol), monoazahemin reconstituted myoglobin showed a more negative redox potential than native myoglobin in a 50 mM bis-Tris buffer solution (pH 6.5), suggesting that for both native and reconstituted myoglobins the heme environment including proximal histidine as an axial ligand of the redox center plays an important role in determining the redox potential. Also, a unique electrochemical response of cyano-monoazahemin reconstituted myoglobin was demonstrated.     

Iron hemiporphycene as a functional prosthetic group for myoglobin

The iron complex of hemiporphycene, a molecular hybrid of porphyrin with porphycene, was incorporated into the apomyoglobin pocket to examine ligand binding ability of the iron atom in the novel porphyrinoid. Apomyoglobin was successfully coupled with a stoichiometric amount of ferric hemiporphycene to afford the reconstituted myoglobin equipped with the iron coordination structure of native protein. Cyanide, imidazole, and fluoride coordinated to the ferric protein with affinities comparable with those for native myoglobin. The ferrous myoglobin was functionally active to bind O(2) and CO reversibly at pH 7.4 and 20 degrees C. The O(2) affinity is 12-fold higher than that of native myoglobin while the CO affinity is slightly lower, suggesting decreased discrimination between O(2) and CO in the heme pocket. The functional anomaly was interpreted to reflect increased sigma-bonding character in the Fe(II)-O(2) bond. In contrast with 6-coordinate native NO protein, the NO myoglobin containing ferrous hemiporphycene is in a mixed 5- and 6-coordinate state. This observation suggests that the in-plane configuration of the iron atom in hemiporphycene is destabilized by NO. Influence of the core deformation was also detected with both the infrared absorption for the ferrous CO derivative and electron paramagnetic resonance for ferric imidazole complex. Anomalies in the ferric and ferrous derivatives were ascribed to the modified iron-N(pyrrole) interactions in the asymmetric metallo core of hemiporphycene.     

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.     

Reductive activation of dioxygen by a myoglobin reconstituted with a flavohemin

We successfully converted myoglobin, an oxygen-storage hemoprotein, into an oxygen-activating hemoprotein like cytochrome P450s by replacing the native hemin with the artificially created flavohemin. The reconstituted myoglobin, rMb(1), was chacterized by ESI-TOF-mass, UV-vis, and fluorescence spectra. The 1H NMR spectrum of cyanomet rMb(1) indicates that two hemin conformers are present in a ratio of 1:1. Upon the addition of NADH to the buffer solution of rMb(1) in the presence of SOD and catalase, the oxymyoglobin was rapidly formed. As compared with the formation of the oxygenated native myoglobin in the presence of 10-N-(acetylaminoethyl)isoalloxazine, the rate constant of the oxyheme formation in rMb(1) is 6 times larger. This is because the flavin covalently linked to the terminal heme propionate functions as an effective mediator of an electron transfer from NADH to the hemin in rMb(1). Furthermore, rMb(1) shows the deformylation activity, when 2-phenylpropionaldehyde (2-PPA) was employed as a substrate. This result indicates that the oxyheme is reductively activated to Fe(III)-peroxoanion (Fe(III)-O22-). The result in this report is the first example of the activation of dioxygen by myoglobin. This study shows the utility of the replacement of the native hemin with a chemically modified one for the functionalization of myoglobin.     

Role of heme types in heme-copper oxidases: effects of replacing a heme b with a heme o mimic in an engineered heme-copper center in myoglobin

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.     

Functional evaluation of iron oxypyriporphyrin in protein heme pocket

The iron complex of oxypyriporphyrin, a porphyrinoid containing a keto-substituted pyridine, was coupled with apomyoglobin. The reconstituted ferric myoglobin was found to be five-coordinate without iron-bound water molecules. The anionic ligands such as CN (-) and N 3 (-) bound the myoglobin with high affinities, while neutral imidazole did not. The IR observation indicated that the azide complex was pure high-spin, although the corresponding native protein was in the spin-state equilibrium. The reduced myoglobin was five-coordinate but exhibited no measurable affinity for O 2. The affinity for CO was lowered down to 1/2400 as compared with native myoglobin. These anomalies were ascribed to the deformation in the iron coordination core after the replacement of one of the four pyrroles with a larger pyridine ring. The ligand binding analyses for the ferric and ferrous myoglobin suggest that the proximal histidine pulls the iron atom from the deformed core to reduce the interaction between the iron and exogenous ligands. Similarity of the reconstituted myoglobin with guanylate cyclase, a NO-responsive signaling hemoprotein, was pointed out.     

Potential energy surface and molecular dynamics of MbNO: existence of an unsuspected FeON minimum

Ligands such as CO, O(2), or NO are involved in the biological function of myoglobin. Here we investigate the energetics and dynamics of NO interacting with the Fe(II) heme group in native myoglobin using ab initio and molecular dynamics simulations. At the global minimum of the ab initio potential energy surface (PES), the binding energy of 23.4 kcal/mol and the Fe-NO structure compare well with the experimental results. Interestingly, the PES is found to exhibit two minima: There exists a metastable, linear Fe-O-N minimum in addition to the known, bent Fe-N-O global minimum conformation. Moreover, the T-shaped configuration is found to be a saddle point, in contrast to the corresponding minimum for NO interacting with Fe(III). To use the ab initio results for finite temperature molecular dynamics simulations, an analytical function was fitted to represent the Fe-NO interaction. The simulations show that the secondary minimum is dynamically stable up to 250 K and has a lifetime of several hundred picoseconds at 300 K. The difference in the topology of the heme-NO PES from that assumed previously (one deep, single Fe-NO minimum) suggests that it is important to use the full PES for a quantitative understanding of this system. Why the metastable state has not been observed in the many spectroscopic studies of myoglobin interacting with NO is discussed, and possible approaches to finding it are outlined.     

A comparative study of O2, CO and CN binding to heme IX protein models

Parametrization of a molecular-mechanics program to include terms specific for five- and six-coordinate transition metal complexes results in computer-simulated structures of heme complexes. The principal new feature peculiar to five and six coordination is a term that measures the effect of electron-pair repulsion modified by the ligand electronegativity and takes into account the different structural possibilities. The model system takes into account the structural differences of the fixing centre in the haemoglobin subunits. The customary proximal histidine is added. The prosthetic group heme IX is wholly considered in our model. The calculations show clearly that certain conformations are much more favourable that others for fixing O2. From the O2 binding in haemoglobin, myoglobin and simple Fe porphyrin models it is concluded that the bent O2 ligand is best viewed as bound superoxide O2-. Axial ligands are practically free-rotating. A small modification of the model in both crystal and protein matrix affects the orientation of the ligands in experimental systems.     

Genetic engineering of the heme pocket in human serum albumin: modulation of O2 binding of iron protoporphyrin IX by variation of distal amino acids

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly: (i) Ile-142-->His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161-->Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O2 binding equilibrium. The coordination structures and ligand (O2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2. In marked contrast, HSA(HL/L185N)-heme showed very high O2 binding affinity (P1/2O2 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O2 binding affinity (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.     

Dioxygenation of human serum albumin having a prosthetic heme group in a tailor-made heme pocket

Human serum albumin (HSA) is the most abundant plasma protein in our bloodstream and serves as a transporter for small hydrophobic molecules such as fatty acids, bilirubin, and steroids. Hemin dissociated from methemoglobin is also bound within a narrow D-shaped cavity in subdomain IB of HSA. In terms of the general hydrophobicity of the alpha-helical pocket, HSA potentially has features similar to the heme-binding site of myoglobin (Mb) or hemoglobin (Hb). However, the reduced ferrous HSA-heme complex is immediately oxidized by O2, because HSA lacks the proximal histidine that enables the heme group to bind O2. In this paper, we report the introduction of a proximal histidine into the subdomain IB of HSA by site-directed mutagenesis to construct a tailor-made heme pocket (I142H/Y161L), which allows a reversible O2 binding to the prosthetic heme group. Laser flash photolysis experiments revealed that this artificial hemoprotein appears to have two different geometries of the axial-imidazole coordination, and these two species (I and II) showed rather low O2 binding affinities (P1/2O2 = 18 and 134 Torr) relative to those of Mb and Hb.     

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.     

Gaseous myoglobin ions stored at greater than 300 K

Multiply-charged myoglobin ions retaining the prosthetic heme group have been formed by electrospray, injected into a quadrupole ion trap, and stored for up to one second prior to mass analysis. Collisional activation experiments indicate that these ions readily fragment into the charged heme group and the complementary apomyoglobin ion. No fragmentation is observed, however, upon ion storage in the presence of a neutral bath gas at 1 × 10^?3 torr for up to one second. The significance of this observation is that these non-covalently-bound ions, in which both the heme group and the polypeptide carry charge, are kinetically stable for over one second at room temperature and, perhaps, at higher temperatures. This suggests that other biologically relevant ions derived using electrospray and bound by non-covalent interactions can be studied using the various tools available with ion storage mass spectrometers and by other techniques that employ relatively high pressure environments for the study of gaseous ions.     

Oxygen‐binding Protein Fiber and Microgel: Supramolecular Myoglobin–Poly(acrylate) Conjugates

A supramolecular conjugate of myoglobin (Mb) and water‐soluble poly(acrylate), (PA_5k and PA_25k, where 5k and 25k represent the molecular weight of the polymers, respectively), is constructed on the basis of a noncovalent heme‐heme pocket interaction. The modified heme with an amino group linked to the terminus of one of the heme‐propionates is coupled to the side‐chain carboxyl groups of poly(acrylate) activated by N‐hydroxysuccinimide. The ratios of the heme‐modified monomer unit and the unmodified monomer unit (m:n) in the polymer chains of Heme‐PA_5k and Heme‐PA_25k were determined to be 4.5:95.5 and 3.1:96.9, respectively. Subsequent addition of apoMb to the conjugated polymers provides Mb‐connected fibrous nanostructures confirmed by atomic force microscopy. A mixture of the heme‐modified polymer and dimeric apomyoglobin as a cross‐linker forms a microgel in which the reconstituted myoglobin retains its native exogenous ligand binding activity.     

Stepwise refolding of Acid-denatured myoglobin: Evidence from electrospray mass spectrometry

Application of electrospray mass spectrometry (ES/MS) to a protein refolding study was demonstrated. Acid denaturation of equine myoglobin was reversed by adding various amounts of ammonium hydroxide to the protein that was unfolded in 10% acetic acid. The protein refolding process was followed by ES/MS, in which both the changes in the protein charge-state distribution and mass were monitored. The ES/MS results show that the pH-dependent renaturation of the acid-denatured myoglobin is stepwise, consisting of two major steps. The unfolded polypeptide chain first refolds to establish a compact nativelike structure, without the assistance of the heme prosthetic group. The newly formed binding cavity then retains the heme group by noncovalent interactions. It is also shown that inclusion of a stabilizing buffer, such as ammonium acetate, in the protein solution is greatly beneficial to the ES/MS detection of intact noncovalent globin/heme complex.     

Heme protein oxygen affinity regulation exerted by proximal effects

Heme proteins are found in all living organisms and are capable of performing a wide variety of tasks, requiring in many cases the binding of diatomic ligands, namely, O(2), CO, and/or NO. Therefore, subtle regulation of these diatomic ligands' affinity is one of the key issues for determining a heme protein's function. This regulation is achieved through direct H-bond interactions between the bound ligand and the protein, and by subtle tuning of the intrinsic heme group reactivity. In this work, we present an investigation of the proximal regulation of oxygen affinity in Fe(II) histidine coordinated heme proteins by means of computer simulation. Density functional theory calculations on heme model systems are used to analyze three proximal effects: charge donation, rotational position, and distance to the heme porphyrin plane of the proximal histidine. In addition, hybrid quantum-classical (QM-MM) calculations were performed in two representative proteins: myoglobin and leghemoglobin. Our results show that all three effects are capable of tuning the Fe-O(2) bond strength in a cooperative way, consistently with the experimental data on oxygen affinity. The proximal effects described herein could operate in a large variety of O(2)-binding heme proteins-in combination with distal effects-and are essential to understand the factors determining a heme protein's O(2) affinity.     

Molecular dynamics study on the solvent dependent heme cooling following ligand photolysis in carbonmonoxy myoglobin

The time scale and mechanism of vibrational energy relaxation of the heme moiety in myoglobin was studied using molecular dynamics simulation. Five different solvent models, including normal water, heavy water, normal glycerol, deuterated glycerol and a nonpolar solvent, and two forms of the heme, one native and one lacking acidic side chains, were studied. Structural alteration of the protein was observed in native myoglobin glycerol solution and native myoglobin water solution. The single-exponential decay of the excess kinetic energy of the heme following ligand photolysis was observed in all systems studied. The relaxation rate depends on the solvent used. However, this dependence cannot be explained using bulk transport properties of the solvent including macroscopic thermal diffusion. The rate and mechanism of heme cooling depends upon the detailed microscopic interaction between the heme and solvent. Three intermolecular energy transfer mechanisms were considered: (i) energy transfer mediated by hydrogen bonds, (ii) direct vibration-vibration energy transfer via resonant interaction, and (iii) energy transfer via vibration-translation or vibration-rotation interaction, or in other words, thermal collision. The hydrogen bond interaction and vibration-vibration interaction between the heme and solvent molecules dominates the energy transfer in native myoglobin aqueous solution and native myoglobin glycerol solutions. For modified myoglobin, the vibration-vibration interaction is also effective in glycerol solution, different from aqueous solution. Thermal collisions form the dominant energy transfer pathway for modified myoglobin in water solution, and for both native myoglobin and modified myoglobin in a nonpolar environment. For native myoglobin in a nonpolar solvent solution, hydrogen bonds between heme isopropionate side chains and nearby protein residues, absent in the modified myoglobin nonpolar solvent solution, are key interactions influencing the relaxation pathways.