Showing papers on "Myoglobin published in 2012"

Biomimetic Catalysis of a Porous Iron-Based Metal–Metalloporphyrin Framework

Abstract: A porous metal–metalloporphyrin framework, MMPF-6, based upon an iron(III)-metalated porphyrin ligand and a secondary binding unit of a zirconium oxide cluster was constructed; MMPF-6 demonstrated interesting peroxidase activity comparable to that of the heme protein myoglobin as well as exhibited solvent adaptability of retaining the peroxidase activity in an organic solvent.

Nitrite Regulates Hypoxic Vasodilation via Myoglobin-Dependent Nitric Oxide Generation

Abstract: Background—Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·. Methods and Results—We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP...

Significant increase of oxidase activity through the genetic incorporation of a tyrosine-histidine cross-link in a myoglobin model of heme-copper oxidase

Abstract: Heme-copper oxidase (HCO) performs efficient four-electron reduction of oxygen to water without releasing toxic, reactive oxygen species (ROS). Essential for this function is a post-translationally modified histidine–tyrosine cross-link (Tyr-His) in its heme a3/CuB oxygen reduction center. Through the genetic incorporation of the Tyr-His ligand and CuB site into myoglobin, we recapitulated important features of HCO into this small soluble protein, which exhibits selective O2 reduction activity while generating less than 6% ROS, at more than 1000 turnovers. These results support that Tyr-His crosslink is indeed important for HCO function, and creates the exciting opportunity to rapidly evolve better HCO model proteins to achieve higher activity and selectivity, which may be suitable as alternatives to precious metal catalyst in fuel cells.

Transition Metals in Biochemistry

Abstract: 1 The Role of Transition Metal Ions in Biological Oxidation and Related Processes.- 1. Transition Metal Ions.- 2. Prosthetic Groups.- 3. Equilibrium Considerations in Reactions of Transition Metals.- a) Site Stability: Equivalent Ligands, Chelation, and Other Factors.- b) Electron Transfer and Redox Potentials.- 4. Molecular Functions of Proteins Containing Transition Metal Ion Prosthetic Groups.- a) Oxygen Carriage.- b) Oxygen Utilization.- c) Hydroperoxide Reactions.- d) Electron Transfer, Protein to Protein.- e) Hydrogen Atom Transfer from Substrate to Coenzyme or Substrate.- f) Nitrogen Utilization.- g) Hydrogen Utilization.- h) Hydrogen Peroxide Formation from Superoxide Radicals.- i) Methyl Transfer and Internal Hydrogen Transfer.- j) Utilization, Production, and Transfer of Ammonia.- k) Oxygen Evolution.- 5. The Role Which a Transition Metal Ion Plays in the Function of a Protein.- 6. Experimental Methods.- a) Isolation and Identification of Prosthetic (Nonprotein) Ligands.- b) X-ray Diffraction from Protein Single Crystals.- c) Electron Paramagnetic Resonance and Related Measurements.- d) Optical Measurements.- e) Measurements Utilizing Nuclei.- 7. Some Aspects of the Role of the Polypeptide in the Functioning of Proteins Containing Transition Metal Ions.- 2 Metal Coordination in Proteins.- 1. Ligands.- 2. The Established Coordination in Several Proteins.- a) Insulin.- b) Carboxypeptidase.- c) Myoglobin and Hemoglobin.- d) Myohemerythrin and Hemerythrin.- e) Cytochrome c.- f) Cytochrome b5.- g) Copper, Zinc Superoxide Dismutase.- h) Rubredoxin.- i) Ferredoxin and High Potential Iron-Sulfur Protein (HiPiP).- 3. Covalency.- 4. Some Aspects of Differences in Heme Binding.- 3 Copper.- 1. Cupric Peptides.- 2. EPR of Cupric Peptides and Related Complexes.- 3. The Blue Proteins.- 4. Magnetic and Optical Properties of Quantum Mechanical Models of the Cupric Ion.- 5. "Nonblue" Coordination in Copper Proteins.- 4 Heme Iron.- 1. Valence and Spin States of Iron.- 2. Magnetic Susceptibility.- 3. Valence State Determination.- 4. Optical Properties.- 5. Spin State Equilibria.- 6. Influences of Symmetry upon the Energy Levels of Low- and High-Spin States.- 7. Ligand Hyperfine Effects in Ferric Hemeproteins.- 8. Iron Hyperfine Effects.- 9. Modified Hemes.- 10. Photodissociation and Recombination.- 5 Nonheme Iron and Molybdenum.- 1. Iron Storage and Transport Proteins.- 2. Iron-Sulfur Proteins.- 3. Molybdenum.- 6 Electronic Structures and Properties.- 1. Atomic Orbitals.- 2. Spin States.- 3. Transition Metal Ions.- 4. Ligands and Molecular Orbitals.- 5. Absorption of Light.- 6. Interaction of Transition-Metal Ions with an Applied Magnetic Field.- 7. Magnetic Interactions of the Metal Electrons with Nuclei in the Coordination Sphere.- 8. Optical Activity.- References.

Functional differentiation of myoglobin isoforms in hypoxia-tolerant carp indicates tissue-specific protective roles

Abstract: Because of a recent whole genome duplication, the hypoxia-tolerant common carp and goldfish are the only vertebrates known to possess two myoglobin (Mb) paralogs. One of these, Mb1, occurs in oxida...

A Comparative Study of Tricarbonylmanganese Photoactivatable CO Releasing Molecules (PhotoCORMs) by Using the Myoglobin Assay and Time‐Resolved IR Spectroscopy

Abstract: Tricarbonylmanganese(I) complexes of the ligands tris(imidazol-4-yl)phosphane (4-tipH), tris(1,4-diisopropylimidazol-2-yl)phosphane (2-tipiPr2), tris(pyridin-2-yl)phosphane (tpp) and tris(N-methylimidazol-2-yl)carbinol (2-ticNMe) were prepared. These act as N,N,N tripodal chelators. The solid-state structure of Mn(CO)3(tpp)]OTf was determined by X-ray diffraction. The potential of these complexes to act as photoactivatable CO-releasing molecules (PhotoCORMs) was studied with the UV/Vis spectroscopy-based myoglobin assay as well as by time-resolved IR spectroscopy. Within the series of compounds prepared, the steric bulk of the imidazolyl groups seems to significantly influence the CO-release kinetics and stoichiometry when using the myoglobin assay. In contrast, the time-resolved IR data suggest release of all carbonyl ligands upon irradiation. This effect points to a much closer association of myoglobin and PhotoCORMs than previously thought and will require further investigation.

Mapping molecular flexibility of proteins with site-directed spin labeling: a case study of myoglobin.

Abstract: Site directed spin labeling (SDSL) has potential for mapping protein flexibility under physiological conditions. The purpose of the present study was to explore this potential using 38 singly spin-labeled mutants of myoglobin distributed throughout the sequence. Correlation of the EPR spectra with protein structure provides new evidence that the site dependent variation in lineshape, and hence motion of the spin label, is due largely to differences in mobility of the helical backbone in the ns time range. Fluctuations between conformational substates, typically in the μs-ms time range, are slow on the EPR time scale and the spectra provide a snapshot of conformational equilibria frozen in time as revealed by multiple components in the spectra. A recent study showed that osmolyte perturbation can positively identify conformational exchange as the origin of multicomponent spectra (Lopez et al. (2009), Protein Sci. 18, 1637). In the present study this new strategy is employed in combination with lineshape analysis and pulsed-EPR interspin distance measurements to investigate the conformation and flexibility of myoglobin in three folded and partially folded states. The regions identified to be in conformational exchange in the three forms agree remarkably well with those assigned by NMR, but the faster time scale of EPR allows characterization of localized states not detected in NMR. Collectively, the results suggest that SDSL-EPR and osmolyte perturbation provide a facile means for mapping the amplitude of fast backbone fluctuations and for detecting sequences in slow conformational exchange in folded and partially folded protein sequences.

Hyper-thermal stability and unprecedented re-folding of solvent-free liquid myoglobin

Abstract: Isolating solvent effects by studying proteins in a liquid phase devoid of solvent has not been previously possible because freeze-dried protein solids do not melt but thermally degrade. Herein we circumvent this problem by modifying the interactions between myoglobin molecules via a polymer-surfactant coronal layer to produce a solvent-free liquid phase that is thermally stable over a wide temperature range. Using high-resolution synchrotron radiation circular dichroism and UV-Vis spectroscopies we determine the temperature-dependent structure and re-folding behaviour of cationized myoglobin under solvent-free conditions, and show that dehydration and subsequent melting of the nanoconstruct has no significant effect on the protein secondary structure at room temperature. Significantly, the solvent-free liquid myoglobin molecules exhibit hyper-thermophilic behaviour and can be reversibly re-folded by cooling from 155 °C. We attribute the abnormally high thermal stability and persistence of protein folding to entropic contributions associated with macromolecular crowding and confinement, and propose that re-folding in the absence of a solvent shell is facilitated by the configurational flexibility and molecular interactivity of the polymer surfactant coronal layer.

Structural and oxygen binding properties of dimeric horse myoglobin

Abstract: Myoglobin (Mb) stores dioxygen in muscles, and is a fundamental model protein widely used in molecular design. The presence of dimeric Mb has been known for more than forty years, but its structural and oxygen binding properties remain unknown. From an X-ray crystallographic analysis at 1.05 A resolution, we found that dimeric metMb exhibits a domain-swapped structure with two extended α-helices. Each new long α-helix is formed by the E and F helices and the EF-loop of the original monomer, and as a result the proximal and distal histidines of the heme originate from different protomers. The heme orientation in the dimer was in the normal mode as in the monomer, but regulated faster from the reverse to normal orientation. The dimer possessed the oxygen binding property, although it exhibited a slightly higher oxygen binding affinity (∼1.4 fold) compared to the monomer and showed no cooperativity for oxygen binding. The oxygen binding rate constant (kon) of the dimer ((14.0 ± 0.7) × 106 M−1 s−1) was similar to that of the monomer, whereas the oxygen dissociation rate constant (koff) of the dimer (8 ± 1 s−1) was smaller than that of the monomer (12 ± 1 s−1). We attribute the similar kon values to their active site structures being similar, whereas the faster regulation of the heme orientation and the smaller koff in the dimer are presumably due to the slight change in the active site structure and/or more rigid structure compared to the monomer. These results show that domain swapping may be a new tool for protein engineering.

Effects of Fiber Type and Size on the Heterogeneity of Oxygen Distribution in Exercising Skeletal Muscle

Abstract: The process of oxygen delivery from capillary to muscle fiber is essential for a tissue with variable oxygen demand, such as skeletal muscle. Oxygen distribution in exercising skeletal muscle is regulated by convective oxygen transport in the blood vessels, oxygen diffusion and consumption in the tissue. Spatial heterogeneities in oxygen supply, such as microvascular architecture and hemodynamic variables, had been observed experimentally and their marked effects on oxygen exchange had been confirmed using mathematical models. In this study, we investigate the effects of heterogeneities in oxygen demand on tissue oxygenation distribution using a multiscale oxygen transport model. Muscles are composed of different ratios of the various fiber types. Each fiber type has characteristic values of several parameters, including fiber size, oxygen consumption, myoglobin concentration, and oxygen diffusivity. Using experimentally measured parameters for different fiber types and applying them to the rat extensor digitorum longus muscle, we evaluated the effects of heterogeneous fiber size and fiber type properties on the oxygen distribution profile. Our simulation results suggest a marked increase in spatial heterogeneity of oxygen due to fiber size distribution in a mixed muscle. Our simulations also suggest that the combined effects of fiber type properties, except size, do not contribute significantly to the tissue oxygen spatial heterogeneity. However, the incorporation of the difference in oxygen consumption rates of different fiber types alone causes higher oxygen heterogeneity compared to control cases with uniform fiber properties. In contrast, incorporating variation in other fiber type-specific properties, such as myoglobin concentration, causes little change in spatial tissue oxygenation profiles.

Hydroxyl radical-modified fibrinogen as a marker of thrombosis: the role of iron.

Abstract: Excessive free iron in blood and in organ tissues (so called iron overload) has been observed in degenerative diseases such as atherosclerosis, cancer, neurological, and certain autoimmune diseases, in which fibrin-like deposits are also found Although most of the body iron is bound to hemoglobin and myoglobin in a divalent ferrous form, a certain amount of iron exists in blood as a trivalent (ferric) ion This particular chemical state of iron has been shown to be toxic to the human body when not controlled by endogenous and/or dietary chelating agents Experiments described in this paper show for the first time that ferric ions (Fe3+) can generate hydroxyl radicals without participation of any redox agent, thus making it a special case of the Fenton reaction Ferric chloride was also demonstrated to induce aggregation of purified fibrinogen at the same molar concentrations that were used for the generation of hydroxyl radicals Iron-aggregated fibrinogen, by contrast to native molecule, could not be di

In the face of hypoxia: myoglobin increases in response to hypoxic conditions and lipid supplementation in cultured Weddell seal skeletal muscle cells

Abstract: SUMMARY A key cellular adaptation to diving in Weddell seals is enhanced myoglobin concentrations in their skeletal muscles, which serve to store oxygen to sustain a lipid-based aerobic metabolism. The aim of this study was to determine whether seal muscle cells are inherently adapted to possess the unique skeletal muscle adaptations to diving seen in the whole animal. We hypothesized that the seal skeletal muscle cells would have enhanced concentrations of myoglobin de novo that would be greater than those from a C 2 C 12 skeletal muscle cell line and reflect the concentrations of myoglobin observed in previous studies. In addition we hypothesized that the seal cells would respond to environmental hypoxia similarly to the C 2 C 12 cells in that citrate synthase activity and myoglobin would remain the same or decrease under hypoxia and lactate dehydrogenase activity would increase under hypoxia as previously reported. We further hypothesized that β-hydroxyacyl CoA dehydrogenase activity would increase in response to the increasing amounts of lipid supplemented to the culture medium. Our results show that myoglobin significantly increases in response to environmental hypoxia and lipids in the Weddell seal cells, while appearing similar metabolically to the C 2 C 12 cells. The results of this study suggest the regulation of myoglobin expression is fundamentally different in Weddell seal skeletal muscle cells when compared with a terrestrial mammalian cell line in that hypoxia and lipids initially prime the skeletal muscles for enhanced myoglobin expression. However, the cells need a secondary stimulus to further increase myoglobin to levels seen in the whole animal.

Localized Hydration in Lyophilized Myoglobin by Hydrogen–Deuterium Exchange Mass Spectrometry. 2. Exchange Kinetics

Abstract: Solid-state hydrogen–deuterium exchange with mass spectrometric analysis (ssHDX) is a promising method for characterizing proteins in amorphous solids. Though analysis of HDX kinetics is informative and well-established in solution, application of these methods to solid samples is complicated by possible heterogeneities in the solid. The studies reported here provide a detailed analysis of the kinetics of hydration and ssHDX for equine myoglobin (Mb) in solid matrices containing sucrose or mannitol. Water sorption was rapid relative to ssHDX, indicating that ssHDX kinetics was not limited by bulk water transport. Deuterium uptake in solids was well-characterized by a biexponential model; values for regression parameters provided insight into differences between the two solid matrices. Analysis of the widths of peptide mass envelopes revealed that, in solution, an apparent EX2 mechanism prevails, consistent with native conformation of the protein. In contrast, in mannitol-containing samples, a smaller non-...

Synthesis, Structures, and CO Releasing Properties of two Tricarbonyl Manganese(I) Complexes

Abstract: [MnBr(CO)5] reacts with one equivalent each of 1, 3, 5-triaza-7-phosphaadamantane (PTA) and sodium diethyldithiocarbamate to afford the yellow MnI complex fac-[Mn(S2CNEt2)(PTA)(CO)3]. Similarly, the same MnI precursor reacts with the sodium salt ofL-histidine to give the tricarbonyl manganese(I) complex fac-[Mn(his)(CO)3] in good yield as a yellow solid. Both compounds were spectroscopically and structurally characterised. The histidinato complex is soluble in water and its aqueous solutions are stable for more than 24 h. The CO releasing properties of the histidinato complex were studied with the myoglobin assay and establish this compound as a novel PhotoCORM, which liberates one of the three carbonyl ligands upon irradiation.

Reversible and irreversible conformational transitions in myoglobin: role of hydrated amino acid ionic liquid.

Abstract: Hydrated phenylalanine ionic liquid (Phe-IL) has been used to solubilize myoglobin (Mb). Structural stability of Mb in Phe-IL analyzed using fluorescence and circular dichroism spectroscopy shows that for low levels of hydration of Phe-IL there is a large red shift in the fluorescence emission wavelength and the protein transforms to complete β sheet from its native helical conformation. Rehydration or dilution reverses the β sheet to an α helix which on aging organizes to micrometer-sized fibrils. At concentrations higher than 200 μM, the protein changes from β to a more random coiled structure. Organization of the protein in Phe-IL in a Langmuir film at the air/water interface has been investigated using the surface pressure–molecular area isotherm and shows nearly the same surface tension for both pure Mb and Mb in Phe-IL. Scanning electron microscopy of the films of Mb in Phe-IL transferred using the Langmuir–Blodgett film technique show layered morphology. This study shows that the conformation of Mb...

Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability Using Mass Spectrometry

Abstract: Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein–ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C2 proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin–haptoglobin (Hb–Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a Kd value of 0.24 nM for the Hb–Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated...

Antioxidant activity of flavonoids evaluated with myoglobin method

Abstract: Antioxidant activities of four flavonoids (rutin, quercetin, luteolin, and kaempferol) and two non-flavonoids (chlorogenic acid and pyrocatechol) against four reactive oxygen species (ROS) have been measured with a myoglobin method developed by our group. The myoglobin method uses the absorbance changes of myoglobin (a probe molecule) due to the reaction with the ROS as an indicator for the antioxidant activity measurement. Myoglobin protective ratio (MPR) was defined to express the antioxidant activities of the specimens. Antioxidant activities against hypochlorite ion, hydroxyl radical, peroxyl radical, and peroxynitrite were measured with the myoglobin method. The antioxidant activities were comprehensively evaluated by plotting MPR against four ROS and vitamin C equivalent concentration evaluated by DPPH quenching method in 5-axe cobweb charts. The four flavonoids show a very similar pattern in the 5-axe cobweb charts, while the patterns of two non-flavonoids are quite different from that of the flavonoids. This procedure combining the myoglobin method with the cobweb charts is useful in the evaluation of antioxidant activities of plant-derived food, and also can be extended to monitor antioxidant condition of media for plant cell cultures.

Engineering Tyrosine-Based Electron Flow Pathways in Proteins: The Case of Aplysia Myoglobin

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

The effect of point mutation on the equilibrium structural fluctuations of ferric Myoglobin

Abstract: The ultrafast equilibrium fluctuations of the FeIII-NO complex of a single point mutation of Myoglobin (H64Q) have been studied using Fourier Transform 2D-IR spectroscopy. Comparison with data from wild type Myoglobin (wt-Mb) shows the presence of two conformational substates of the mutant haem pocket where only one exists in the wild type form. One of the substates of the mutant exhibits an almost identical NO stretching frequency and spectral diffusion dynamics to wt-Mb while the other is distinctly different in both respects. The remarkably contrasting dynamics are largely attributable to interactions between the NO ligand and a nearby distal side chain which provides a basis for understanding the roles of these side chains in other ferric haem proteins.

Synthesis of iron nanoparticles from hemoglobin and myoglobin

Abstract: Stable iron nanoparticles have been synthesized from naturally occurring and abundant Fe-containing bio-precursors, namely hemoglobin and myoglobin. The formation of stable iron nanoparticles was achieved through a one-pot, single-phase chemical reduction approach. The reduction of iron ions present in the bio-precursors was carried out at room temperature and avoids the use of harsh chemical reagents. The size distribution of the product falls into the narrow 2?5?nm range and the particles were found to be crystalline. This method can be a valuable synthetic approach for producing bio-conjugated nanoparticle systems for biological applications.

Protein Conformation-Controlled Rebinding Barrier of NO and Its Binding Trajectories in Myoglobin and Hemoglobin at Room Temperature

Abstract: The effect of the solvent viscosity on the dynamics of NO rebinding to myoglobin (Mb) and hemoglobin (Hb) was examined by femtosecond (fs) time-resolved vibrational spectroscopy after photodeligation of NO from MbNO and HbNO in various viscous solutions at 283 K using a 580 nm excitation pulse. The rebinding kinetics of NO to both Mb and Hb were nonexponential, but their dependence on the solvent viscosity was different. The rate of NO rebinding to Mb increased with increasing solution viscosity, which was achieved by increasing the glycerol content in glycerol/water mixture. In contrast, the rate of NO rebinding to Hb was independent of the solution viscosity but faster than the fastest rate of NO rebinding observed in Mb. The dynamics of conformational relaxation of the protein after deligation were also measured by probing the evolution of the amide band. The effect of the solvent viscosity on the kinetics of conformational relaxation in both proteins was also quite different. The conformational relaxa...

Bridging the gap between chemistry, physiology, and evolution: quantifying the functionality of sperm whale myoglobin mutants.

Abstract: This work merges a large set of previously reported thermochemical data for myoglobin (Mb) mutants with a physiological model of O 2 -transport and -storage. The model allows a quantification of the functional proficiency of myoglobin (Mb) mutants under various physiological conditions, i.e. O 2 -consumption rate resembling workload, O 2 partial pressure resembling hypoxic stress, muscle cell size, and Mb concentration, resembling different organism-specific and compensatory variables. We find that O 2 -storage and -transport are distinct functions that rank mutants and wild type differently depending on O 2 partial pressure. Specifically, the wild type is near-optimal for storage at all conditions, but for transport only at severely hypoxic conditions. At normoxic conditions, low-affinity mutants are in fact better O 2 -transporters because they still have empty sites for O 2 , giving rise to a larger [MbO 2 ] gradient (more varying saturation curve). The distributions of functionality reveal that many mutants are near-neutral with respect to function, whereas only a few are strongly affected, and the variation in functionality increases dramatically at lower O 2 pressure. These results together show that conserved residues in wild type (WT) Mb were fixated under a selection pressure of low P O2 .

Relationship between oxygen affinity and autoxidation of myoglobin.

Abstract: Studies using myoglobins reconstituted with a variety of chemically modified heme cofactors revealed that the oxygen affinity and autoxidation reaction rate of the proteins are highly correlated to each other, both decreasing with decreasing the electron density of the heme iron atom. An Fe(3+)-O(2)(-)-like species has been expected for the Fe(2+)-O(2) bond in the protein, and the electron density of the heme iron atom influences the resonance process between the two forms. A shift of the resonance toward the Fe(2+)-O(2) form results in lowering of the O(2) affinity due to an increase in the O(2) dissociation rate. On the other hand, a shift of the resonance toward the Fe(3+)-O(2)(-)-like species results in acceleration of the autoxidation through increasing H(+) affinity of the bound ligand.

Absorption Band III Kinetics Probe the Picosecond Heme Iron Motion Triggered by Nitric Oxide Binding to Hemoglobin and Myoglobin

Abstract: To study the ultrafast movement of the heme iron induced by nitric oxide (NO) binding to hemoglobin (Hb) and myoglobin (Mb), we probed the picosecond spectral evolution of absorption band III (∼760 nm) and vibrational modes (iron–histidine stretching, ν4 and ν7 in-plane modes) in time-resolved resonance Raman spectra. The time constants of band III intensity kinetics induced by NO rebinding (25 ps for hemoglobin and 40 ps for myoglobin) are larger than in Soret bands and Q-bands. Band III intensity kinetics is retarded with respect to NO rebinding to Hb and to Mb. Similarly, the ν(Fe–His) stretching intensity kinetics are retarded with respect to the ν4 and ν7 heme modes and to Soret absorption. In contrast, band III spectral shift kinetics do not coincide with band III intensity kinetics but follows Soret kinetics. We concluded that, namely, the band III intensity depends on the heme iron out-of-plane position, as theoretically predicted (Stavrov, S. S.Biopolymers 2004, 74, 37−40).

Myoglobin functioning as cytochrome P450 for biosensing of 2,4-dichlorophenol

Abstract: The interactions between 2,4-dichlorophenol (2,4-DCP) and myoglobin immobilized by agarose hydrogel on the surface of a glassy carbon electrode were explored by cyclic voltammetry. 2,4-DCP coordinating with the heme of myoglobin induces a negative shift in the formal potential of myoglobin without occurrence of a catalytic reaction in anaerobic solution. However, the immobilized myoglobin functioned as cytochrome P450 under the catalytic pathways of C-hydroxylation of 2,4-DCP in an air-saturated solution. The plot of current against 2,4-DCP concentration shows a linear relationship in the range of 12.5–208 μM. The limit of detection is calculated to be 2.06 μM. UV spectra confirm that 2,4-DCP interacts with the amino-acid residues of myoglobin as well as Mb-heme. This type of study can provide important insights into the mechanisms involved in the interaction of hemoproteins with chlorophenols. The methods possess potential applications in biotechnology and biosensors.