Heme Propionate Side Chains Research Articles

Hemoproteins Reconstituted with Artificial Metal Complexes as Biohybrid Catalysts.

In nature, heme cofactor-containing proteins participate not only in electron transfer and O2 storage and transport but also in biosynthesis and degradation. The simplest and representative cofactor, heme b, is bound within the heme pocket via noncovalent interaction in many hemoproteins, suggesting that the cofactor is removable from the protein, leaving a unique cavity. Since the cavity functions as a coordination sphere for heme, it is of particular interest to investigate replacement of native heme with an artificial metal complex, because the substituted metal complex will be stabilized in the heme pocket while providing alternative chemical properties. Thus, cofactor substitution has great potential for engineering of hemoproteins with alternative functions. For these studies, myoglobin has been a focus of our investigations, because it is a well-known oxygen storage hemoprotein. However, the heme pocket of myoglobin has been only arranged for stabilizing the heme-bound dioxygen, so the structure is not suitable for activation of small molecules such as H2O2 and O2 as well as for binding an external substrate. Thus, the conversion of myoglobin to an enzyme-like biocatalyst has presented significant challenges. The results of our investigations have provided useful information for chemists and biologists. Our own efforts to develop functionalized myoglobin have focused on the incorporation of a chemically modified cofactor into apomyoglobin in order to (1) construct an artificial substrate-binding site near the heme pocket, (2) increase cofactor reactivity, or (3) promote a new reaction that has never before been catalyzed by a native heme enzyme. In pursuing these objectives, we first found that myoglobin reconstituted with heme having a chemically modified heme-propionate side chain at the exit of the heme pocket has peroxidase activity with respect to oxidation of phenol derivatives. Our recent investigations have succeeded in enhancing oxidation and oxygenation activities of myoglobin as well as promoting new reactions by reconstitution of myoglobin with new porphyrinoid metal complexes. Incorporation of suitable metal porphyrinoids into the heme pocket has produced artificial enzymes capable of efficiently generating reactive high valent metal-oxo and metallocarbene intermediates to achieve the catalytic hydroxylation of C(sp3)-H bonds and cyclopropanation of olefin molecules, respectively. In other efforts, we have focused on nitrobindin, an NO-binding hemoprotein, because aponitrobindin includes a β-barrel cavity, which provides a robust structure highly similar to that of the native holoprotein. It was expected that the aponitrobindin would be suitable for development as a protein scaffold for a metal complex. Recently, it was confirmed that several organometallic complexes can bind to this scaffold and function as catalysts promoting hydrogen evolution or C-C bond formation. The hydrophobic β-barrel structure plays a significant role in substrate binding as well as controlling the stereoselectivity of the reactions. Furthermore, these catalytic activities and stereoselectivities are remarkably improved by mutation-dependent modifications of the cavity structure for the artificial cofactor. This Account demonstrates how apoproteins of hemoproteins can provide useful protein scaffolds for metal complexes. Further development of these concepts will provide a useful strategy for generation of robust and useful artificial metalloenzymes.

Cofactor-specific covalent anchoring of cytochrome b562on a single-walled carbon nanotube by click chemistry

Redox-active cytochromeb562with a tethered azide group on the heme propionate side chain is covalently linked to an acetylene moiety introduced on the sidewall of a single-walled carbon nanotube by copper-catalyzed click chemistry.

The crystal structure of isoniazid-bound KatG catalase-peroxidase from Synechococcuselongatus PCC7942.

Isoniazid (INH) is one of the most effective antibiotics against tuberculosis. INH is a prodrug that is activated by KatG. Although extensive studies have been performed in order to understand the mechanism of KatG, even the binding site of INH in KatG remains controversial. In this study, we determined the crystal structure of KatG from Synechococcus elongatus PCC7942 (SeKatG) in a complex with INH at 2.12-Å resolution. Three INH molecules were bound to the molecular surface. One INH molecule was bound at the entrance to the ε-edge side of heme (designated site 1), another was bound at the entrance to the γ-edge side of heme (site 2), and another was bound to the loop structures in front of the heme propionate side chain (site 3). All of the interactions between KatG and the bound INH seemed to be weak, being mediated mainly by van der Waals contacts. Structural comparisons revealed that the identity and configuration of the residues in site 1 were very similar among SeKatG, Burkholderia pseudomallei KatG, and Mycobacterium tuberculosis KatG. In contrast, sites 2 and 3 were structurally diverse among the three proteins. Thus, site 1 is probably the common KatG INH-binding site. A static enzymatic analysis and thermal shift assay suggested that the INH-activating reaction does not proceed in site 1, but rather that this site may function as an initial trapping site for the INH molecule. Database: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession number 3WXO.

Preparation and Physicochemical Properties of Supramolecular Hemoprotein Assembly Via Heme-Heme Pocket Interaction

Protoheme IX is the simplest and most well-known iron porphyrin in nature and acts as a cofactor in many hemoproteins such as hemoglobin, myoglobin, cytochrome P450, and horseradish peroxidase. The cofactor, heme b, is bound in the heme pocket via coordination and multiple noncovalent interactions. Therefore, heme b is released from the heme pocket upon acidic condition to yield the apoprotein. The apoprotein is capable of binding heme b again at neutral pH value and then the reconstituted protein is available. To functionalize a hemoprotein, it will be very effective for replacing native heme with an appropriate heme analog. Our group has recently focused on the following three methods to prepare an artificial prosthetic group.1) (i) Introduction of functional groups at the terminal of the heme-propionate side chains. (ii) Modification of the framework of the prosthetic group. (iii) Replacement of a metal substitution in the core of the porphyrin framework. In our previous work, we prepared several types of supramoelcular hemoprotein assembies via interprotein heme–heme pocket interaction by method (i) vide supra.2) We first prepared a heme moiety with a maleimide group at the terminal of one of the heme-propionate side chains. The heme moiety was then introduced to a cysteine residue positioned on the surface of an apocytochrome b 562 mutant (H63C) via a covalent linkage. The heme moiety-linked apoprotein provided a linear hemoprotein assembly determined by an AFM technique.3) The AFM image clearly demonstrates the 1D protein fiber including more than 100 proteins on a graphite substrate. Next, we tried to immobilize the hemoprotein polymers onto a gold surface, gold electrode or gold nanoparticle. Particularly, the heme-attached gold surface provided the hemoprotein assemblies with the number of 6–8 protein layers on the electrode upon the addition of the heme-linked apoproteins.4) The assembly structure on the electrode was mainly analyzed by the AFM technique and QCM measurement. Furthermore, the immobilization of zinc cytochrome b 562 polymer on a gold electrode gave efficient photocurrent generation in the presence of methyl viologen as an electron mediator. In addition, it is found that heme-immobilized gold nanoparticles provide a unique self-assembly upon the addition of apohemoprotein dimer.5) The supramolecular porphyrin array mediated by hemoprotein matrix serves as a new way to create bionanomaterials. For example, we have prepared a myoglobin–quantum dot conjugate by a heme-immobilized CdTe particle.6) In this presentation, we report the construction of several supramolecular hemoprotein assemblies in a solution and on a metal surface. 1) T. Hayashi, In Handbook of Porphyrin Science; K. M. Kadish, K. M. Smith, R. Guilard, Eds.; World Scientific publishing: Singapore, 2010; Vol. 5, pp 1–69.2) K. Oohora, A. Onoda, T. Hayashi, Chem. Commun., 48, 11714–11726 (2012) (Feature Article). 3) H. Kitagishi, K. Oohora, H. Yamaguchi, H. Sato, T. Matsuo, A. Harada, T. Hayashi, J. Am. Chem. Soc., 129, 10326–10327 (2007). 4) A. Onoda, Y. Kakikura, T. Uematsu, S. Kuwabata, T. Hayashi, Angew. Chem. Int. Ed., 51, 2628–2631 (2012). 5) A. Onoda, Y. Ueya, T. Sakamoto, T. Uematsu, T. Hayashi, Chem. Commun., 46, 9107–9109 (2010).6) A. Onoda, T. Himiyama, K. Ohkubo, S. Fukuzumi, T. Hayashi, Chem. Commun., 48, 8054–8056 (2012).

DNA-Binding Hemoproteins Tethering Polyamine Interface

Abstract Artificial prosthetic groups tethering a polyamine moiety at the terminal of two peripheral heme–propionate side chains as a molecular interface were inserted into apocytochrome b562 to provide positively charged reconstituted hemoproteins, which exhibit a strong binding with double-stranded DNA.

A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating the Access of Water Molecules to the Substrate-Binding Site

Cytochrome P450cam is a heme-containing enzyme which catalyzes hydroxylation of d-camphor. The heme is bound in the heme pocket via noncovalent interactions, where two heme-propionate side chains interact with Arg, His, and/or Asp residues. To understand the role of the heme-7-propionate side chain, we prepared reconstituted P450cam with an artificial one-legged heme which has a methyl group at the position of the 7-propionate. Removal of 7-propionate dramatically decreases the d-camphor affinity by 3 orders of magnitude relative to that of the wild-type enzyme, and spectroscopic data indicate that 74% of the ferric P450cam exhibits a low-spin state owing to water molecule occupancy in the substrate-binding site under the normal assay conditions. Thus, the monooxygenase activity of the reconstituted protein is remarkably low due to the decrease in the rate of the first electron transfer from reduced putidaredoxin, whereas 87% of oxidized NADH was utilized to produce 5-hydroxy-d-camphor without any significant uncoupling reactions. X-ray structural analysis of the reconstituted enzyme reveals a novel water array extending from the substrate-binding site to bulk solvent through the position occupied by 7-propionate. This water array appears without causing any major changes in the protein structure with the notable exception of conformational changes occurring at Asp297 and Gln322 residues. We propose that the 7-propionate forms a barrier against entry of bulk water molecules and therefore in combination with Asp297, Arg299, and Gln322 plays an essential role in the process of elimination of the substrate-binding site water cluster which occurs upon d-camphor binding.

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".

Evaluation of the Functional Role of the Heme-6-propionate Side Chain in Cytochrome P450cam

Cytochrome P450cam (P450cam) binds a protoheme IX as a prosthetic group via noncovalent interactions. Heme-6-propionate, one of the two heme-propionate side chains, forms hydrogen-bonding interactions with Arg112 and other hydrophilic amino acid residues. Here, we demonstrate the structural and functional roles of the 6-propionate side chain in P450cam using a reconstituted protein with 6-depropionate-6-methylated protoheme IX (one-legged heme). The spectroscopic data and the enzymatic activities reveal that removal of the 6-propionate has no clear influence on the enzyme property. The rate of electron transfer from putidaredoxin (Pdx), a natural redox partner, to P450cam was not significantly changed, whereas, the removal of the 6-propionate decreased the affinity of Pdx by 3.5-fold supporting the proposed role of Arg112 as the essential constituent of the Pdx binding site. Resonance Raman experiments indicate that removal of the 6-propionate weakens the Fe−S bond strength. The X-ray structure of the rec...

Structure and Ligand Binding Properties of Myoglobins Reconstituted with Monodepropionated Heme: Functional Role of Each Heme Propionate Side Chain,

Two heme propionate side chains, which are attached at the 6 and 7 positions of the heme framework, are linked with Arg45 and Ser92, respectively, in sperm whale myoglobin. To evaluate the role of each propionate, two kinds of one-legged hemins, 6-depropionated and 7-depropionated protohemins, were prepared and inserted into the apomyoglobin to yield two reconstituted proteins. Structural data of the reconstituted myoglobins were obtained via an X-ray crystallographic analysis at a resolution of 1.1-1.4 A and resonance Raman spectroscopy. It was found that the lack of the 6-propionate reduces the number of hydrogen bonds in the distal site and clearly changes the position of the Arg45 residue with the disrupting Arg45-Asp60 interaction. In contrast, the removal of the 7-propionate does not cause a significant structural change in the residues of the distal and proximal sites. However, the resonance Raman studies suggested that the coordination bond strength of the His93-Fe bond for the protein with the 7-depropionated protoheme slightly increases compared to that for the protein with the native heme. The O2 and CO ligand binding studies for the reconstituted proteins with the one-legged hemes provide an important insight into the functional role of each propionate. The lack of the 6-propionate accelerates the O2 dissociation by ca. 3-fold compared to those of the other reconstituted and native proteins. The lack of the 7-propionate enhances the CO affinity by 2-fold compared to that of the protein with the native heme. These results indicate that the 6-propionate clearly contributes to the stabilization of the bound O2, whereas the 7-propionate plays an important role in the regulation of the Fe-His bond.

Time-resolved Raman evidence for energy ‘funneling’ through propionate side chains in heme ‘cooling’ upon photolysis of carbonmonoxy myoglobin

The heme ‘cooling’ following CO photolysis was investigated with picosecond time-resolved anti-Stokes Raman spectroscopy on modified myoglobins (Mb), in which the 6- or 7-propionate is selectively replaced by a methyl group. The time constants of population decay of vibrationally excited states for two modified Mbs became significantly larger compared with those of native Mb. This is the first experimental evidence for appreciable contribution of each heme-propionate side chain to the vibrational energy transfer from the heme to the surroundings.

YcdB from Escherichia coli Reveals a Novel Class of Tat-dependently Translocated Hemoproteins

The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin Fe(III)-protoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.

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.

Chemical Properties of Sperm Whale Myoglobins Reconstituted with Monopropionate Hemins

Abstract The binding behavior of two heme-propionate side chains in sperm whale myoglobin was evaluated using artificially created hemins, 6-methyl-7-propionate- and 6-propionate-7-methyl-protohemin IX. From the thermodynamic study of the hemin binding to apomyoglobin, it was found that two heme-propionates clearly contribute to the stabilization of the hemin in the protein matrix.

Enhancement of enzymatic activity for myoglobins by modification of heme-propionate side chains

The modification of myoglobin is an attractive process not only for understanding its molecular mechanism but also for engineering the protein function. The strategy of myoglobin functionalization can be divided into at least two approaches: site-directed mutagenesis and reconstitution with a non-natural prosthetic group. The former method enables us to mainly modulate the physiological function, while the latter has the advantage of introducing a new function on the protein. Particularly, replacement of the native hemin with an artificially created hemin having hydrophobic moieties at the terminal of the heme-propionate side chains serves as an appropriate substrate-binding site near the heme pocket, and consequently enhances the peroxidase and peroxygenase activities for the reconstituted myoglobin. In addition, the incorporation of the synthetic hemin bearing modified heme-propionates into an appropriate apomyoglobin mutant drastically enhances the peroxidase activity. In contrast, to convert myoglobin into a cytochrome P450 enzyme, a flavin moiety as an electron transfer mediator was introduced at the terminal of the heme-propionate side chain. The flavomyoglobin catalyzes the deformylation of 2-phenylpropanal in the presence of NADH under aerobic conditions through the peroxoanion formation from the oxygenated species. In addition, modification of the heme-propionate side chains has an significant influence on regulating the reactivity of the horseradish peroxidase. Furthermore, the heme-propionate side chain can form a metal binding site with a carboxylate residue in the heme pocket. These studies indicate that modification of the heme-propionate side chains can be a new and effective way to engineer functions for the hemoproteins.

Enhancement of Peroxygenase Activity of Horse Heart Myoglobin by Modification of Heme-propionate Side Chains

Abstract A myoglobin reconstituted with an artificially created heme having two benzene moieties bound at the terminal of two heme-propionates shows a clear enhancement in peroxygenase activity toward thioether and styrene oxidations due to the expanding hydrophobic heme pocket.

Contribution of heme-propionate side chains to structure and function of myoglobin: chemical approach by artificially created prosthetic groups

Horse heart myoglobin was reconstituted with mesohemin derivatives methylated at the 6- or 7-position to evaluate the role of the heme-6-propionate or heme-7-propionate side chain in the protein. The association and dissociation of the O2 binding for the deoxymyoglobin with 6-methyl-7-propionate mesoheme are clearly accelerated. Furthermore, the myoglobin with 6-methyl-7-propionate mesoheme shows fast autoxidation from oxymyoglobin to metmyoglobin compared to the myoglobin with 6-propionate-7-methyl heme and the reference protein. These results indicate the 6-propionate plays an important physiological role in the stabilization of oxymyoglobin because of the formation of a salt-bridge with the Lys45. The acceleration of CO binding rate is observed for the myoglobin with 6-propionate-7-methyl mesoheme, suggesting that the replacement of the 7-propionate with a methyl group has an influence on the His93–heme iron coordination. The structural perturbation of His93 imidazole was also supported by 1H NMR spectra of cyanide and deoxy forms of the myoglobin with 6-propionate-7-methyl mesoheme. Thus, it is found that the 7-propionate regulates the hydrogen-bonding network and His93–heme iron coordination in the proximal site.

Structure and stability effects of the mutation of glycine 34 to serine in Rhodobacter capsulatus cytochrome c(2).

Gly 34 and the adjacent Pro 35 of Rhodobacter capsulatus cytochrome c(2) (or Gly 29 and Pro 30 in vertebrate cytochrome c) are highly conserved side chains among the class I c-type cytochromes. The mutation of Gly 34 to Ser in Rb. capsulatus cytochrome c(2) has been characterized in terms of physicochemical properties and NMR in both redox states. A comparison of the wild-type cytochrome c(2), the G34S mutation, and the P35A mutation is presented in the context of differences in chemical shifts, the differences in NOE patterns, and structural changes resulting from oxidation of the reduced cytochrome. G34S is substantially destabilized relative to wild-type (2.2 kcal/mol in the oxidized state) but similarly destabilized relative to P35A. Nevertheless, differences in terms of the impact of the mutations on specific structural regions are found when comparing G34S and P35A. Although available data indicates that the overall secondary structure of G34S and wild-type cytochrome c(2) are similar, a number of both perturbations of hydrogen bond networks and interactions with internal waters are found. Thus, the impact of the mutation at position 35 is propagated throughout the cytochrome but with alterations at defined sites within the molecule. Interestingly, we find that the substitution of serine at position 34 results in a perturbation of the heme beta meso and the methyl-5 protons. This suggests that the hydroxyl and beta carbon are positioned away from the solvent and toward the heme. This has the consequence of preferentially stabilizing the oxidized state in G34S, thus, altering hydrogen bond networks which involve the heme propionate, internal waters, and key amino acid side chains. The results presented provide important new insights into the stability and solution structure of the cytochrome c(2).

Cytochrome b−245 of the Neutrophil Superoxide-generating System Contains Two Nonidentical Hemes: POTENTIOMETRIC STUDIES OF A MUTANT FORM OF gp91phox

Analysis of potentiometric titrations of the cytochrome b-245 from a X+ chronic granulomatous disease patient with an Arg54 --> Ser mutation in gp91phox indicates that the mutant form of the cytochrome contains two nonidentical hemes with midpoint potentials of Em7 = -220 and Em7 = -300 mV. In the light of this information, reanalysis of redox titrations of wild-type cytochrome b-245 implies that it probably also contains two separate heme centers with midpoint potentials of Em7 = -225 and Em7 = -265 mV. The effect of the Arg54 --> Ser substitution is to reduce the midpoint potential of one of the heme centers by approximately 35 mV and suggests possible interaction between Arg54 and a heme propionate side chain.

Assignment of heme resonances in the 1H NMR spectrum of oxidized Desulfovibrio vulgaris (Hildenborough) cytochrome c3

A combination of 2D COSY and NOESY experiments and 1D nuclear Overhauser effect measurements have allowed an extensive assignment of the hyperfine-shifted heme resonances in the downfield region of the 1H NMR spectrum of the fully oxidized tetraheme cytochrome c 3 from Desulfovibrio vulgaris, Hildenborough. Fifteen of the sixteen heme methyl groups were identified and assigned, some on a tentative basis. Most of the one-proton paramagnetic resonances were demonstrated to arise from the α-CH 2 groups of the heme propionate side chains, and from the β-CH 2 groups of the iron-binding axial histidines. Assignment of methyl resonances to individual hemes was achieved by reference to previous calculations of electron density distributions on the four hemes of the highly homologous Miyazaki protein obtained by NMR data ( Biochemistry, 29 (1990) 2257). The methyl hyperfine shift patterns are apparently different for the individual hemes. Despite the complexity of the system due to interheme interactions, we have determined that these patterns appear to be influenced by the orientation of the imidazole planes of the iron-binding axial histidines, as found for simpler monoheme proteins.

Heme sulfuric anhydrides as soybean leghemoglobin structure probes

Mesoheme monosulfuric anhydride reacts at three distinct sites in soybean apoleghemoglobin a, at lysine-6, lysine-19 and lysine-57, the last one being the major site of reaction. The heme peptides obtained from thermolytic and pronase hydrolysates of the anhydride-leghemoglobin a were purified and correlated with the known amino acid sequence of the protein. Mesoheme bissulfuric anhydride also reacts with soybean apoleghemoglobin a giving a complex mixture of hemepeptides after hydrolysis with pronase. The visible spectrum of anhydride leghemoglobin is that of low spin heme. This suggests that anhydride leghemoglobin has a conformation with a covalent attachment via propionic acid side chain to lysine-57 and the sixth coordination position of the heme iron occupied by the distal histidine at position 61. Native leghemoglobin is assumed to exist in a similar type of configuration at low temperature, but with the heme propionate side chain being involved in a salt bridge with lysine-57.