Essential Amino Acid Residues Controlling the Unique Regioselectivity of Heme Oxygenase in Pseudomonas aeruginosa

Heme oxygenase (HO), an amphipathic microsomal protein, catalyzes the oxygen-dependent degradation of heme (iron-protoporphyrinIX) to alpha-biliverdin, CO, and free iron ion. Interestingly, all of HO regiospecifically oxidize the alpha-meso position of the heme to form alpha-biliverdin isomer while nonenzymatic heme degradation forms all four possible alpha-, beta-, gamma-, delta-biliverdin isomers at nearly identical yield. Recently, an interesting example has been found in HO (PigA) of the Gram-negative bacterium Pseudomonas aeruginosa, which does not produce alpha-biliverdin at all, but forms the mixture of beta- and gamma-biliverdins at a ratio of 3:7. While studying the mechanism of the unique regioselectivty of PigA, we found essential amino acid residues, Lys34, Lys132, and Phe189, controlling the unique regioselectivity of PigA. In this communication, we show that Lys34 and Lys132 are essential amino acid residues to hold the rotated heme in the active site of PigA via hydrogen-bonding interaction with the heme propionate and that Phe189 controls the product ratio of beta- and delta-biliverdins via steric interaction with heme substituents. These interactions place the beta- or delta-meso position of the heme at the oxidation site of PigA, leading to the unique regioselectivity.     

Quantum mechanical/molecular mechanical study of mechanisms of heme degradation by the enzyme heme oxygenase: the strategic function of the water cluster

Heme degradation by heme oxygenase (HO) enzymes is important in maintaining iron homeostasis and prevention of oxidative stress, etc. In response to mechanistic uncertainties, we performed quantum mechanical/molecular mechanical investigations of the heme hydroxylation by HO, in the native route and with the oxygen surrogate donor H2O2. It is demonstrated that H2O2 cannot be deprotonated to yield Fe(III)OOH, and hence the surrogate reaction starts from the FeHOOH complex. The calculations show that, when starting from either Fe(III)OOH or Fe(III)HOOH, the fully concerted mechanism involving O-O bond breakage and O-C(meso) bond formation is highly disfavored. The low-energy mechanism involves a nonsynchronous, effectively concerted pathway, in which the active species undergoes first O-O bond homolysis followed by a barrier-free (small with Fe(III)HOOH) hydroxyl radical attack on the meso position of the porphyrin. During the reaction of Fe(III)HOOH, formation of the Por+*FeIV=O species, compound I, competes with heme hydroxylation, thereby reducing the efficiency of the surrogate route. All these conclusions are in accord with experimental findings (Chu, G. C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M. J. Biol. Chem. 1999, 274, 21319). The study highlights the role of the water cluster in the distal pocket in creating "function" for the enzyme; this cluster affects the O-O cleavage and the O-Cmeso formation, but more so it is responsible for the orientation of the hydroxyl radical and for the observed alpha-meso regioselectivity of hydroxylation (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). Differences/similarities with P450 and HRP are discussed.     

13C NMR spectroscopy of core heme carbons as a simple tool to elucidate the coordination state of ferric high-spin heme proteins

Evidence is presented demonstrating that the magnitudes of the 13C chemical shifts originating from heme meso carbons provide a straightforward diagnostic tool to elucidate the coordination state of high-spin heme proteins and enzymes. Pentacoordinate high-spin heme centers exhibit 13C meso shifts centered at approximately 250 ppm, whereas their hexacoordinate counterparts exhibit 13C shifts centered at approximately -80 ppm. The relatively small spectral window (400 to -100 ppm) covering the meso-13C shifts, the relatively narrow lines of these resonances, and the availability of biosynthetic methods to prepare 13C-labeled heme (Rivera, M.; Walker, F. A. Anal. Biochem. 1995, 230, 295-302) make this approach practical. The theoretical basis for the distinct chemical shifts observed for meso carbons from hexacoordinate high-spin hemes relative to their pentacoordinate counterparts are now well understood (Cheng, R.-J.; Chen, P. Y.; Lovell, T.; Liu, T.; Noodleman, L.; Case, D. A. J. Am. Chem. Soc. 2003, 125, 6774-6783), which indicates that the magnitude of the meso-carbon chemical shifts can be used as a simple and reliable diagnostic tool for determining the coordination state of the heme active sites, independent of the nature of the proximal ligand. Proof of the principle for the 13C NMR spectroscopic approach is demonstrated using hexa- and pentacoordinate myoglobin. Subsequently, 13C NMR spectroscopy has been used to unambiguously determine that a recently discovered heme protein from Shigella dysenteriae (ShuT) is pentacoordinate.     

Modulation of ligand-field parameters by heme ruffling in cytochromes c revealed by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectra of variants of Hydrogenobacter thermophilus cytochrome c(552) (Ht c-552) and Pseudomonas aeruginosa cytochrome c(551) (Pa c-551) are analyzed to determine the effect of heme ruffling on ligand-field parameters. Mutations introduced at positions 13 and 22 in Ht c-552 were previously demonstrated to influence hydrogen bonding in the proximal heme pocket and to tune reduction potential (E(m)) over a range of 80 mV [Michel, L. V.; Ye, T.; Bowman, S. E. J.; Levin, B. D.; Hahn, M. A.; Russell, B. S.; Elliott, S. J.; Bren, K. L. Biochemistry 2007, 46, 11753-11760]. These mutations are shown here to also increase heme ruffling as E(m) decreases. The primary effect on electronic structure of increasing heme ruffling is found to be a decrease in the axial ligand-field term Δ/λ, which is proposed to arise from an increase in the energy of the d(xy) orbital. Mutations at position 7, previously demonstrated to influence heme ruffling in Pa c-551 and Ht c-552, are utilized to test this correlation between molecular and electronic structure. In conclusion, the structure of the proximal heme pocket of cytochromes c is shown to play a role in determining heme conformation and electronic structure.     

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.     

Marked difference in the electronic structure of cyanide-ligated ferric protoglobins and myoglobin due to heme ruffling

Electron paramagnetic resonance experiments reveal a significant difference between the principal g values (and hence ligand-field parameters) of the ferric cyanide-ligated form of different variants of the protoglobin of Methanosarcina acetivorans (MaPgb) and of horse heart myoglobin (hhMb). The largest principal g value of the ferric cyanide-ligated MaPgb variants is found to be significantly lower than for any of the other globins reported so far. This is at least partially caused by the strong heme distortions as proven by the determination of the hyperfine interaction of the heme nitrogens and mesoprotons. Furthermore, the experiments confirm recent theoretical predictions [Forti, F.; Boechi, L., Bikiel, D., Martí, M.A.; Nardini, M.; Bolognesi, M.; Viappiani, C.; Estrin, D.; Luque, F. J. J. Phys. Chem. B2011, 115, 13771-13780] that Phe(G8)145 plays a crucial role in the ligand modulation in MaPgb. Finally, the influence of the N-terminal 20 amino-acid chain on the heme pocket in these protoglobins is also proven.     

Alkylation of heme by the antimalarial drug artemisinin

The peroxide function of artemisinin has been activated by iron(II)-heme generated in situ from iron(III)-protoporphyrin-IX and glutathione, a biologically relevant reductant. In mild conditions, this reaction produced a high yield (85%) of heme derivatives alkylated at alpha-, beta-, and delta-meso positions by a C4-centered radical derived from artemisinin.     

Assignment of the ferriheme resonances of the high-spin forms of nitrophorins 1 and 4 by 1H NMR spectroscopy: comparison to structural data obtained from X-ray crystallography

In this work, we report the assignment of the majority of the ferriheme resonances of high-spin nitrophorins (NPs) 1 and 4 and compare them to those of NP2, published previously. It is found that the structures of the ferriheme complexes of NP1 and NP4, in terms of the orientation of the histidine imidazole ligand, can be described with good accuracy by NMR techniques and that the angle plot proposed previously for the high-spin form of the NPs (Shokhireva, T. Kh.; Shokhirev, N. V.; Walker, F. A. Biochemistry 2003, 42, 679-693) describes the angle of the effective nodal plane of the axial histidine imidazole in solution. There is an equilibrium between the two heme orientations (A and B), which depends on the heme cavity shape, which can be altered by mutation of amino acids with side chains (phenyl vs tyrosyl) near the potential position where a heme vinyl group would be in one of the isomers. The A:B ratio can be much more accurately measured by NMR spectroscopy than by X-ray crystallography.     

Radical energies and the regiochemistry of addition to heme groups. Methylperoxy and nitrite radical additions to the heme of horseradish peroxidase

The heme of hemoproteins, as exemplified by horseradish peroxidase (HRP), can undergo additions at the meso carbons and/or vinyl groups of the electrophilic or radical species generated in the catalytic oxidation of halides, pseudohalides, carboxylic acids, aryl and alkyl hydrazines, and other substrates. The determinants of the regiospecificity of these reactions, however, are unclear. We report here modification of the heme of HRP by autocatalytically generated, low-energy NO2* and CH3OO* radicals. The NO2* radical adds regioselectively to the 4- over the 2-vinyl group but does not add to the meso positions. Reaction of HRP with tert-BuOOH does not lead to heme modification; however, reaction with the F152M mutant, in which the heme vinyls are more sterically accessible, results in conversion of the heme 2-vinyl into a 1-hydroxy-2-(methylperoxy)ethyl group [-CH(OH)CH2OOCH3]. [18O]-labeling studies indicate that the hydroxyl group in this adduct derives from water and the methylperoxide oxygens from O2. Under anaerobic conditions, methyl radicals formed by fragmentation of the autocatalytically generated tert-BuO* radical add to both the delta-meso carbon and the 2-vinyl group. The regiochemistry of these and the other known additions to the heme indicate that only high-energy radicals (e.g., CH3*) add to the meso carbon. Less energetic radicals, including NO2* and CH3OO*, add to heme vinyl groups if they are small enough but do not add to the meso carbons. Electrophilic species such as HOBr, HOCl, and HOSCN add to vinyl groups but do not react with the meso carbons. This meso- versus vinyl-reactivity paradigm, which appears to be general for autocatalytic additions to heme prosthetic groups, suggests that meso hydroxylation of the heme by heme oxygenase occurs by a controlled radical reaction rather than by electrophilic addition.     

Model studies for heme oxygenase-catalyzed porphyrin meso hydroxylation

[structure: see text]. Nonenzymatic model studies based on a porphyrin analogue (2,4-diacetyldeuteroporphyrin) that avoid the steric effect complications of the heme oxygenase active site were carried out to determine the polarity of the ferric hydroperoxide attacking species. Mass spectral and deuterium-labeling experiments indicate that the porphyrin meso positions that are at higher pi-electron densities in ferric 2,4-diacetyldeuteroporphyrin are selectively attacked. This supports an electrophilic aromatic substitution mechanism for the heme oxygenase-catalyzed porphyrin meso hydroxylation.     

Structures of the Heme Acquisition Protein HasA with Iron(III)‐5,15‐Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group

Iron(III)‐5,15‐diphenylporphyrin and several derivatives were accommodated by HasA, a heme acquisition protein secreted by Pseudomonas aeruginosa , despite possessing bulky substituents at the meso position of the porphyrin. Crystal structure analysis revealed that the two phenyl groups at the meso positions of porphyrin extend outside HasA. It was shown that the growth of P. aeruginosa was inhibited in the presence of HasA coordinating the synthetic porphyrins under iron‐limiting conditions, and that the structure of the synthetic porphyrins greatly affects the inhibition efficiency.     

An electroreflectance approach to study out the puzzling state of myoglobin in a DDAB film on a pyrolytic graphite electrode surface

The state of the heme of myoglobin molecules incorporated in a didodecyldimethylammonium bromide (DDAB) film on a pyrolytic graphite electrode was described using the results of electroreflectance measurements. It was found that the hemes are released from the myoglobin molecules. The ER spectrum of PG electrode|Mb–DDAB film was indistinguishable from the spectrum of PG electrode|hemin–DDAB film, even in the presence of NaBr, but clearly different from PG electrode|imidazole-coordinated hemin–DDAB. These results support the claim of de Groot and coworker [M.T. de Groot, M. Merksx, M.T.M. Koper, J. Am. Chem. Soc. 127 (2005) 16224; M.T. de Groot, M. Merkx, M.T.M. Koper, Electrochem. Commun. 8 (2006) 999]. It is likely that DDAB is not a strong inhibitor of imidazole coordination but acts on the protein, resulting in conformational change and the heme release.     

Atropisomers of meso-conjugated uracyl porphyrin derivatives and their assembling structures

[reaction: see text] We have synthesized a 5,15 meso-substituted methyluracyl porphyrin derivative bearing 6-methyluracyl units directly at the meso positions. The atropisomerization was regulated by steric replusion between the methyl substituents. When the atropisomers were mixed with alkylated melamine as a complementary hydrogen-bonding unit, the hydrogen-bonded assemblies were analyzed by diffusion-ordered spectroscopy (DOSY) in solution, which clarified that the alphabeta isomer formed a face-to-face dimer, whereas the alphabeta isomer took a zigzag structure.     

Azide-inhibited bacterial heme oxygenases exhibit an S = 3/2 (dxz,dyz)3(dxy)1(dz2)1 spin state: mechanistic implications for heme oxidation

The azide complexes of heme oxygenase from Pseudomonas aeruginosa (pa-HO) and Neisseriae meningitidis (nm-HO) have been studied with the aid of (1)H and (13)C NMR spectroscopy. These complexes have been shown to exist as an equilibrium mixture of two populations, one exhibiting an S = (1)/(2), (d(xy))(2)(d(xz), d(yz))(3) electron configuration and planar heme and a second with a novel S = (3)/(2), (d(xz), d(yz))(3)(d(xy))(1)(d(z)(2))(1) spin state and nonplanar heme. At physiologically relevant temperatures, the equilibrium shifts in the direction of the population exhibiting the latter electron configuration and nonplanar heme, whereas at temperatures approaching the freezing point of water, the equilibrium shifts in the direction of the population with the former electronic structure and planar heme. These findings indicate that the microenvironment of the distal pocket in heme oxygenase is unique among heme-containing proteins in that it lowers the sigma-donating (field strength) ability of the distal ligand and, therefore, promotes the attainment of heme electronic structures thus far only observed in heme oxygenase. When the field strength of the distal ligand is slightly lower than that of azide, such as OH(-) (J. Am. Chem. Soc. 2003, 125, 11842), the corresponding complex exists as a mixture of populations with nonplanar hemes and electronic structures that place significant spin density at the meso positions. The ease with which these unusual heme electronic structures are attained by heme oxygenase is likely related to activation of meso carbon reactivity which, in turn, facilitates hydroxylation of a meso carbon by the obligatory ferric hydroperoxide intermediate.     

Resonance Raman spectroscopic studies of hydroperoxo-myoglobin at cryogenic temperatures

In agreement with previous reports (Gasyna, Z. FEBS Lett. 1979, 106, 213-218 and Leibl, W.; Nitschke, W.; Huettermann, J. Biochim. Biophys. Acta 1986, 870, 20-30) radiolytically reduced samples of oxygenated myoglobin at cryogenic temperatures have been shown by optical absorption and EPR studies to produce directly the peroxo-bound myoglobin at 77 K. Annealing to temperatures near 185 K induces proton transfer, resulting in the formation of the hydroperoxo heme derivative. Resonance Raman studies of the annealed samples has permitted, for the first time, the direct observation of the key nu(Fe-O) stretching mode of the physiologically important Fe-OOH fragment of this ubiquitous intermediate. The assignment of this mode to a feature appearing at 617 cm(-1) is strongly supported by documentation of a 25 cm(-1) shift to lower energy upon substitution with (18)O(2) and by a 5 cm(-1) shift to lower energy for samples prepared in solutions of deuterated solvent.     

Synthesis of CJ-15,208, a novel κ-opioid receptor antagonist

The tryptophan isomers of the cyclic tetrapeptide CJ-15,208, reported to be a kappa opioid receptor (KOR) antagonist [Saito, T.; Hirai, H.; Kim, Y. J.; Kojima, Y.; Matsunaga, Y.; Nishida, H.; Sakakibara, T.; Suga, O.; Sujaku, T.; Kojima, N. J. Antibiot. (Tokyo)2002, 55, 847-854.], were synthesized to determine the tryptophan stereochemistry in the natural product. A strategy was developed to select linear precursor peptides that favor cyclization using molecular modeling, and optimized cyclization conditions are reported. The optical rotation of the l-Trp isomer is consistent with that of the natural product. Unexpectedly both isomers exhibit similar nanomolar affinity for KOR.     

Distortional Supramolecular Isomers of Polyrotaxane Coordination Polymers: Photoreactivity and Sensing of Nitro Compounds

Distortional isomers, or bond‐stretch isomers, differ only in the length of one or more bonds, which is due to crystallographic disorder in most cases. The term distortional isomerism is introduced to describe the structures of polyrotaxane 2D coordination polymers (CPs) that differ only by the relative positions in the neighboring entangled axles. A large ring and a long spacer ligand in 2D CPs yielded four different supramolecular isomers, of which two have an entangled polyrotaxane structure. One pair of C?C bonds in the spacer ligand is well‐aligned in one isomer and undergoes [2+2] cycloaddition reaction, whereas the other isomer is photoinert. They also have different sensing efficiency for several aromatic nitro compounds. However, both isomers show selective PL quenching for the Brady’s reagent. Structurally similar supramolecular isomers with different photochemical reactivity and sensing abilities appear to be unprecedented.     

Reductive dimerization of alkylidenemalonates using samarium(II) diiodide and 1H-NMR behavior of the dimers, 2,3-diaryl-1,1,4,4-butanetetracarboxylates

Alkylidenemalonates were readily dimerized in the presence of SmI2 to give 2,3-disubstituted 1,1,4,4-butanetetracarboxylates as mixtures of meso and racemic isomers in moderate to good yields. The structure of the less polar isomer of tetraethyl 2,3-diphenyl-1,1,4,4-butanetetracarboxylate was determined by X-ray crystallographic analysis to be the meso form. Characteristic 1H-NMR behavior of the meso and racemic isomers is also discussed.     

Covalent Anchor Positions Play an Important Role in Tuning Catalytic Properties of a Rationally Designed MnSalen-containing Metalloenzyme

Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds, and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over the single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +14% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta- and para- substituted substrates. The 1?Mb(T39C/L72C) showed the highest reactivity (TON up to 2.31 min(-1)) and selectivity (ee% up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.     

Internal electric field in cytochrome C explored by visible electronic circular dichroism spectroscopy

Electronic circular dichroism (ECD) is a valuable tool to explore the secondary and tertiary structure of proteins. With respect to heme proteins, the corresponding visible ECD spectra, which probe the chirality of the heme environment, have been used to explore functionally relevant structural changes in the heme vicinity. While the physical basis of the obtained ECD signal has been analyzed by Woody and co-workers in terms of multiple electronic coupling mechanism between the electronic transitions of the heme chromophore and of the protein (Hsu, M.C.; Woody, R.W. J. Am. Chem. Soc. 1971, 93, 3515), a theory for a detailed quantitative analysis of ECD profiles has only recently been developed (Schweitzer-Stenner, R.; Gorden, J. P.; Hagarman, A. J. Chem. Phys. 2007, 127, 135103). In the present study this theory is applied to analyze the visible ECD-spectra of both oxidation states of three cytochromes c from horse, cow and yeast. The results reveal that both B- and Q-bands are subject to band splitting, which is caused by a combination of electronic and vibronic perturbations. The B-band splittings are substantially larger than the corresponding Q-band splittings in both oxidation states. For the B-bands, the electronic contribution to the band splitting can be assigned to the internal electric field in the heme pocket, whereas the corresponding Q-band splitting is likely to reflect its gradient (Manas, E. S.; Vanderkooi, J. M.; Sharp, K. A. J. Phys. Chem. B 1999, 103, 6344). We found that the electronic and vibronic splitting is substantially larger in the oxidized than in the reduced state. Moreover, these states exhibit different signs of electronic splitting. These findings suggest that the oxidation process increases the internal electric field and changes its orientation with respect to the molecular coordinate system associated with the N-Fe-N lines of the heme group. For the reduced state, we used our data to calculate electric field strengths between 27 and 31 MV/cm for the investigated cytochrome c species. The field of the oxidized state is more difficult to estimate, owing to the lack of information about its orientation in the heme plane. Based on band splitting and the wavenumber of the band position we estimated a field-strength of ca. 40 MV/cm for oxidized horse heart cytochrome c. The thus derived difference between the field strengths of the oxidized and reduced state would contribute at least -55 kJ/mol to the enthalpic stabilization of the oxidized state. Our data indicate that the corresponding stabilization energy of yeast cytochrome c is smaller.