1H NMR study of the effect of heme insertion on the folding of apomyoglobin

NMR signals arising from His EF5 and His GH1 N_ϵH protons of sperm whale myoglobin and apomyoglobin have been assigned, and the protein folding has been studied through the analysis of these signals. His EF5 and His GH1 N_ϵH protons participate in the internal hydrogen bonds at the B–GH and EF–H interfaces, respectively, and their signals are remarkably sensitive to local structural alterations at these sites. The shifts of these signals in alkaline pH condition were only slightly affected by the removal of heme, indicating that the overall protein folding is essentially retained in apoprotein. The line width of His EF5 proton signal, however, increased largely in the spectra of apomyoglobin and this result suggests a conformational lability of the EF–H interface in the absence of heme. Furthermore, the His EF5 proton signal was found to be influenced by not only the orientation of heme relative to the protein, but also by the type of hemin used to reconstitute apomyoglobin. These results clearly demonstrate the presence of a long-range structural correlation between the heme active site and the EF–H interface.     

Construction of glycosylated myoglobin by reconstitutional method

An artificial prosthetic group having beta-galactosyl moieties was synthesized and inserted into sperm whale apomyoglobin to successfully afford a glycosylated myoglobin.     

Resolution of tryptophan-ANS fluorescence energy transfer in apomyoglobin by site-directed mutagenesis

Resonance energy transfer between tryptophanyl residues and the apolar fluorescent dye 1-anilino-8-naphthalene sulfonate (ANS) occurs when the fluorophore is bound to native folded sperm whale apomyoglobin. The individual transfer contribution of the two tryptophanyl residues (W7 and W14, both located on the A-helix of the protein) was resolved by measuring the tryptophan-ANS transfer efficiency for the ANS-apomyoglobin complexes formed by wild-type protein and protein mutants containing one or no tryptophanyl residues, i.e. W7F, W14F and W7YW14F. The transfer efficiency of W14 residue was found to be higher than that of W7, thus indicating that W14 acts as the main energy donor in the ANS-apomyoglobin complex. This suggests that the plane containing the anilinonaphthalene ring of the extrinsic fluorophore has a spatial orientation similar to that of W14 and, hence, to the heme group in the holoprotein.     

Spectroelectrochemistry of heme proteins: effects of active-site heterogeneity on Nernst plots

In order to detect and model the effect of functional chain heterogeneity on Nernst plots for heme proteins, we examined the redox properties of various myoglobins (Mbs) and their mixtures using an improved spectroelectrochemical method. Specific redox responses and formal half potentials (E1/2) were obtained for Aplysia, horse, and sperm whale Mbs, as well as 1:1 mixtures of Mbs consisting of Aplysia/sperm whale, sperm whale/horse, and horse/Aplysia. Linear Nernst plots with slopes near unity were observed for horse, sperm whale, and Aplysia Mbs, with E1/2 values of 14, 19, and 96 mV (vs. NHE) respectively, consistent with previous reports using indirect methods. The Nernst plot responses for mixtures of some of these Mbs allowed us to evaluate and model the non-Nernstian behavior that results from intrinsically different values of E1/2 and from incomplete spectral overlap. The data demonstrate that increasing the E1/2 differences between the components of a Mb mixture increases the changes in shape of the resulting Nernst plots, the dominant effect being a decrease in the observed Nernst coefficient (nNernst). Comparison of Nernst plots for redox data with Hill plots for O2 binding data shows that the redox process is more affected by the structural differences in the distal heme pockets of the Mbs studied than is O2 binding. Similar effects of chain heterogeneity may give rise to disproportionate reductions in the slopes of Nernst and Hill plots for hemoglobins (Hbs). This possibility is discussed in relation to Hbs investigated for redox and O2 binding activity in our laboratories where we find nNernst to be commonly less than nHill over a range of experimental conditions.     

Computer simulations of the refolding of sperm whale apomyoglobin from high-temperature denaturated state

The refolding mechanism of apomyoglobin (apoMb) subsequent to high-temperature unfolding has been examined using computer simulations with atomic level detail. The folding of this protein has been extensively studied experimentally, providing a large database of folding parameters which can be probed using simulations. In the present study, 4-folding trajectories of apoMb were computed starting from coiled structures. A crystal structure of sperm whale myoglobin taken from the Protein Data Bank was used to construct the final native conformation by removal of the heme group followed by energy optimization. The initial unfolded conformations were obtained from high-temperature molecular dynamics simulations. Room-temperature refolding trajectories at neutral pH were obtained using the stochastic difference equation in length algorithm. The folding trajectories were compared with experimental results and two previous molecular dynamics studies at low pH. In contrast to the previous simulations, an extended intermediate with large helical content was not observed. In the present study, a structural collapse occurs without formation of helices or native contacts. Once the protein structure is more compact (radius of gyration<18 A) secondary and tertiary structures appear. These results suggest that apoMb follows a different folding pathway after high-temperature denaturation.     

A Hemin-Analogous Corrphycene Derivative: Suppression of Heme Oxygenase and Reconstitution with Apomyoglobin

Summary. A closely hemin-analogous corrphycene derivative was prepared in good overall yield. By spectroscopic measurements it was shown that it complexes with the stress protein heme oxygenase and apomyoglobin in a similar way as hemin. However, due to its molecular structure it is not attacked by heme oxygenase, but is able to block this enzyme to some degree. In addition, the complex with apomyoglobin displays oxygen and carbon monoxide ligation comparable to myoglobin. These properties make this novel corrphycene derivative a candidate to be used as heme oxygenase blocker or otherwise as a blood pigment substitute.Received July 15, 2003; accepted September 5, 2003 Published online October 23, 2003     

The H93G Myoglobin Cavity Mutant as a Versatile Scaffold for Modeling Heme Iron Coordination Structures in Protein Active Sites and Their Characterization with Magnetic Circular Dichroism Spectroscopy

Preparation of heme model complexes is a challenging subject of long-standing interest for inorganic chemists. His93Gly sperm whale myoglobin (H93G Mb) has the proximal His replaced with the much smaller non-coordinating Gly. This leaves a cavity on the proximal side of the heme into which a wide variety of exogenous ligands can be delivered. The end result is a remarkably versatile scaffold for the preparation of model heme adducts to mimic the heme iron coordination structure of native heme proteins. In this review, we first summarize the quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of the H93G Mb cavity mutant that facilitates the preparation of mixed-ligand derivatives. Then we review our use of magnetic circular dichroism and electronic absorption spectroscopy to characterize nitrogen-, oxygen-, and sulfur-donor-ligated H93G Mb adducts with an emphasis on species not easily prepared by other heme model system approaches and those that serve as spectroscopic models for native heme proteins.     

The correct molecular weight of myoglobin, a common calibrant for mass spectrometry.

Myoglobins from horse heart muscle, horse skeletal muscle and sperm whale are widely used as calibration standards or test compounds for various mass spectrometric methodologies. In all such cases reported in the literature, a molecular weight value is used (16,950.5 and 17,199, respectively) which is based on the assumption that amino acid 122 in this 153 amino-acid-long protein is asparagine, overlooking a published suggestion that it is aspartic acid instead. Since the mass assignment accuracy for matrix-assisted laser desorption mass spectrometry is reported to be +/- 0.01% and for electrospray ionization +/- 0.0025%, and error of one mass unit in approximately 17,000 would be significant. The mass-to-charge ratio of ions of the tryptic peptide encompassing amino acid 122 derived from commercially available horse heart and horse skeletal myoglobins, the apomyoglobin of the latter, and the tryptic and chymotryptic peptide of sperm whale myoglobin proved that in both proteins amino acid 122 is indeed aspartic acid, rather than asparagine. This finding was further confirmed by the collision-induced dissociation spectra of the [M + H]+ ions of the tryptic peptides from the horse myoglobins and the chymotriptic peptide from sperm whale myoglobin. Thus, the correct molecular weight of horse myoglobin is 16,951.49 and that of the sperm whale protein is 17,199.91.     

Structural and functional properties of designed globins

De novo design of artificial proteins is an essential approach to elucidate the principles of protein architecture and to understand specific functions of natural proteins and also to yield novel molecules for medical and industrial aims. We have designed artificial sequences of 153 amino acids to fit the main-chain framework of the sperm whale myoglobin structure based on the knowledge-based energy functions to evaluate the compatibility between protein tertiary structures and amino acid sequences. The synthesized artificial globins bind a single heme per protein molecule as designed, which show well-defined electrochemical and spectroscopic features characteristic of proteins with a low-spin heme. Redox and ligand binding reactions of the artificial heme proteins were investigated and these heme-related functions were found to vary with their structural uniqueness. Relationships between the structural and functional properties are discussed.     

Studies on ligand binding of kidney bean leghemoglobin.

Absorption spectra of different ligand derivative;s of kidney bean leghemoglobin alpha have been recorded. The effect of pH on the absorption spectra of kidney bean leghemoglobin alpha has been studied. The pK of the acid-alkaline transition of the heme-linked water molecule is 8.25 and the pK for the acid dissociation of the heme group is 4.03. Affinities of kidney bean leghemoglobin for two different types of ligands have been studied in comparison with soybean leghemoglobins alpha and c and sperm whale myoglobin. All these leghemoglobins have similar affinities for the small anionic ligand fluoride ion, and they are only slightly more accessible to this ligand than is sperm whale myoglobin. Differences in the primary structure or in conformation of these proteins are reflected in the affinity for the bulky ligand imidazole. The accessibility to imidazole increases in the order sperm whale myoglobin less than soybean Lbalpha less than soybean Lbc less than kidney bean Lbalpha, and also low spin Lbalpha less than high spin Lbalpha. The results are discussed with respect to the amino acid sequences of the leghemoglobins.     

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.     

19F NMR characterization of the thermodynamics and dynamics of the acid-alkaline transition in a reconstituted sperm whale metmyoglobin

A 19F NMR study on the acid-alkaline transition in sperm whale myoglobin reconstituted with a perfluoromethyl heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), demonstrated that the thermodynamics of the transition is predominantly controlled by the stability of acidic form.     

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.     

Nonequilibrium dynamics simulations of nitric oxide release: comparative study of nitrophorin and myoglobin

Nitrophorin 4 (NP4) is a heme protein that reversibly binds nitric oxide (NO), with release rates modulated by pH change. High-resolution structures of NP4 revealed that pH changes and NO binding induce a large conformational rearrangement in two loops that serve to protect the heme-bound NO molecule from solvent. We used extended (110 ns) molecular dynamics simulations of NP4 at pH 5 and pH 7, modeled by selective deprotonation of acidic groups. Conformational and dynamic changes were observed, consistent with those found in the crystal. Further, major solvent movement and NO escape were observed at pH 7, while the ligand remained in the heme binding pocket at pH 5. As a control, we also performed molecular dynamics (MD) simulations of sperm whale myoglobin, where NO migration into the interior cavities of the protein was observed, consistent with previous reports. We constructed a kinetic model of ligand escape to quantitatively relate the microscopic rate constants to the observed rates, and tested the predictions against the experimental data. The results suggest that release rates of diatomic molecules from heme proteins can be varied by several orders of magnitude through modest adjustments in geminate rebinding and gating behavior.     

Equilibrium O2 distribution in the Zn2+-protoporphyrin IX deoxymyoglobin mimic: application to oxygen migration pathway analysis

Proton spin relaxation induced by the triplet ground state of O(2) in the zinc-containing diamagnetic analogue of sperm whale deoxymyoglobin has been measured as a function of oxygen concentration. As no covalent binding of oxygen to the metal occurs in the zinc species, the relaxation effects of O(2) on the protein (1)H resonances arise exclusively via much weaker noncovalent interactions. The relaxation effects at the amide proton sites are found to be highly localized and are derived almost exclusively from O(2) binding at the four previously identified xenon binding sites. Relative binding constants of 1.0, 0.08, 0.07, and 0.23 were determined for the Xe 1, Xe 2, Xe 3, and Xe 4 sites, respectively. In combination with earlier measurements of the kinetics of the heme binding of oxygen, these equilibria measurements enable a more detailed analysis of models characterizing O(2) entry and egress. A correlation is established between the fraction of O(2) which enters the Fe(2+)-binding site via rotation of the distal histidine side chain (so-called "histidine gate") and the experimentally observable O(2) (or CO) lifetime in the Xe 1 site. A physiological role for these secondary oxygen binding sites is proposed in enhancing the efficiency of the O(2) association reaction by rendering more favorable its competition with water binding in the distal heme pocket.     

Ion-ion proton transfer reactions of bio-ions involving noncovalent interactions: Holomyoglobin

Multiply protonated horse skeletal muscle holomyoglobin and apomyoglobin have been subjected to ion-ion proton transfer reactions with anions derived from perfluoro-1,3-dimethylcyclohexane in a quadrupole ion trap operated with helium as a bath gas at 1 mtorr. Neither the apomyoglobin nor holomyoglobin ions show any sign of fragmentation associated with charge state reduction to the 1 + charge state. This is particularly noteworthy for the holomyoglobin ions, which retain the noncovalently bound heme group. For example, no sign of heme loss is associated with charge state reduction from the 9 + charge state of holomyoglobin to the 1 + charge state despite the eight consecutive highly exothermic proton transfer reactions required to bring about this charge change. This result is consistent with calculations that show the combination of long ion lifetime and the high ion-helium collision rate relative to the ion-ion collision rate makes fragmentation unlikely for high mass ions in the ion trap environment even for noncovalently bound complexes of moderate binding strength. The ion-ion proton transfer rates for holo- and apomyoglobin ions of the same charge state also were observed to be indistinguishable, which supports the expectation that ion-ion proton transfer rates are insensitive to ion structure and are determined primarily by the attractive Coulomb field.     

Characterization of conformational changes and noncovalent complexes of myoglobin by electrospray ionization mass spectrometry,circular dichroism and fluorescence spectroscopy

Electrospray ionization mass spectrometry (ESI‐MS) was employed to monitor the heme release and the conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI‐MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study the mechanism of acid‐ and organic solvent‐induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme–protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2–3.6, or solution containing 20–30% acetonitrile or 40–50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI‐MS conditions were established to observe the H₂O‐ and CO‐bound Mb complexes, respectively. H₂O binding to metmyoglobin (17 585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI‐MS. CO binding to Mb (17 595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI‐MS combined with spectroscopic techniques provides a useful means for probing the formation of ligand‐binding complexes and characterizing protein conformational changes. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of an adsorbed reversed-phase packing material for the separation of proteins and peptides

We have described the preparation and chromatographic evaluation of an adsorbed hydrophobic stationary phase suitable for reversed-phase chromatography of proteins and peptides. The synthetic procedure involves three steps: the adsorption of a polyamine to the silica surface; crosslinking of the adsorbed polyamine layer with a bis-phenyl difunctional epoxide; and the benzoylation of the remaining accessible amino groups. Performance of this chromatographic material compared favorably with SynChropak RP-8 silica (SynChrom, Linden, IN, U.S.A.) and was stable to 40% formic acid. Good separations were obtained between the components of sample mixtures containing proteins or the cyanogen bromide fragments of sperm whale myoglobin. However, in both cases, the adsorbed hydrophobic stationary phase was less retentive. Furthermore, this medium exhibited slightly different selectivity. Whereas the heme which was present in the cyanogen bromide digest of myoglobin desorbed as the second peak from the RP-8 column, it eluted last from the adsorbed stationary phase. Comparable performance, acid stability and alternate selectivity suggest that this material is an interesting alternative to organosilane reversed-phase coatings.     

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.     

Dynamic Simulations of the Hydrogen-bondings on the Proximal Side of the Heme in Terms of the ABEEM/MM Method

The structures of sperm whale myoglobin (Mb) and mutants were investigated in terms of the ABEEM/MM method. The molecular dynamic simulations showed that the bifurcated hydrogen-bondings in the proximal side of the heme in Mb were not stable. These simulations indicated that hydrogen-bondings could not determine the overall orientation of imidazole, which could be related to the histidine residue. The amide acids and the bulk of the imidazole can have effects on the flexibility of proximal ligands.