Myoglobin Derivatives Research Articles

Local Structure of Heme-Iron Studied by High-Resolution XANES; Thermal Spin Equilibrium in Myoglobin

The local structure of heme-iron of alkaline met-myoglobin (MbOH) during the thermal spin equilibrium between high and low spin states has been studied by a high-resolution XANES. The spin-state sensitive near edge features have been found in the pre-edge ls-3d transition peak and absorption threshold region. These characteristic features are dependent on the high spin concentration and are related to the d-electron configuration and local structure of heme-iron, i. e. the distortion of octahedral ligand field caused by a displacement of heme-iron from the heme plane. The spin-state sensitive near-edge structures were also found for other myoglobin derivatives and can be used as a spin state marker. From a systematic high resolution XANES study on MbCO, MbCN and oxy-Mb), near-edge features which are sensitive to the angle between the diatomic ligand molecule and heme normal were found. These characteristic features can provide the elaborate local structure of heme-iron such as the displacement of iron atom from the hemeplane or the bond angle of ligand molecule when combined with EXAFS experiments.

Purification to homogeneity and initial physical characterization of secondary amine monooxygenase.

Secondary amine monooxygenase from Pseudomonas aminovorans grown on trimethylamine has been purified 265-fold to apparent homogeneity. The purified enzyme exhibits a specific activity of 14.7 mumol of NADPH oxidized per min per mg of protein, a native molecular weight of 210,000, and nondisulfide-linked subunits of molecular weight 42,000, 36,000, and 24,000, each of which is required for activity. The enzyme is extremely labile during purification; rapid handling and the presence of 5% ethanol are essential to enzyme stability. Storage at 77 K in the presence of NADH (1 mM) also confers considerable stability to the purified enzyme. The heme prosthetic group in the enzyme has been identified as protoporphyrin IX. The quantification of prosthetic group components reveals the presence of 1.6 mol of flavin as FMN, 2.0 mol of heme iron, 4.0 mol of acid-soluble (nonheme) iron, and 3.6 mol of free sulfide/210,000 molecular weight enzyme. Ferric and ferrous-CO secondary amine monooxygenase exhibit electronic absorption spectra that are very similar to those of analogous myoglobin derivatives and, therefore, quite distinct from parallel forms of cytochrome P-450, the most extensively studied heme iron-containing monooxygenase. Like myoglobin and, again, in contrast to P-450, this enzyme forms a very stable dioxygen complex. In fact, it is this oxygen-bound form of the enzyme that is obtained from the purification procedure. Once again, the absorption spectrum of oxygenated secondary amine monooxygenase is nearly identical to that of oxymyoglobin. The spectroscopic similarities between secondary amine monooxygenase and myoglobin suggest the presence of an endogenous histidine fifth ligand to the heme iron of the enzymes.

13C-NMR study of labeled vinyl groups in paramagnetic myoglobin derivatives

The 13C-NMR spectra of high-spin met-aquo myoglobin, spin-equilibrium met-azido myoglobin, low-spin met-cyano myoglobin, deoxy myoglobin and carbonmonoxy myoglobin from sperm whale reconstituted with hemin 13C enriched at both vinyl α or β positions have been recorded. In all cases the labeled vinyl 13C signals are clearly resolved and useful spectra could be obtained within approx. 15 minutes. The decoupling of multiplet structure due to attached proton(s) has led to the specific assignment of vinyl 13C α signals in all paramagnetic derivatives and the 13C β signals in met-cyano myoglobin. In all other cases, the collapse of the proton multiplet structure as a function of 1H decoupling frequency has located, but not assigned, the attached 1H resonance positions which are obscured by the intense diamagnetic envelope in the 1H-NMR spectrum. The resulting vinyl 13C hyperfine shifts follow Curie behavior, and the patterns closely resemble those in the appropriate model complexes in the same oxidation/spin/ligation state, except that the protein exhibits more in-plane asymmetry. The hyperfine shift patterns are indicative of dominant π contact shifts for all ferric complexes. Deoxy myoglobin vinyl 13C and 1H contact shifts provide little evidence for π bonding.

Crystallization and preliminary diffraction data for horse heart metmyoglobin

Reddish-brown crystals of metmyoglobin from horse heart have been obtained by both the hanging drop and batch crystallization methods in the space group P2 1, having a = 64.3 A ̊ , b = 28.9 A ̊ and c = 35.9 A ̊ , with β = 107.1 ° . Morphologically similar crystal forms have been obtained for three derivatives of horse heart myoglobin having modified heme prosthetic groups.

The interaction of radiation-generated radicals with myoglobin in aqueous solution—V. The indirect action of 2-methyl-2-hydroxypropyl radicals on oxymyoglobin

The interaction of radiation-generated 2-methyl-2-hydroxypropyl radicals (derived from t-butyl alcohol) with oxymyoglobin has been examined at pH 7.3. In N 2O-saturated solutions, oxymyoglobin is converted to the ferri and ferryl derivatives of myoglobin; the production of ferrylmyoglobin is essentially eliminated when catalase is present in solution during irradiation. In deaerated solutions containing catalase, oxymyoglobin is converted to both ferro- and ferrimyoglobin during irradiation. When added O 2 is initially present, all compositional changes occur after irradiation; the presence of catalase diminishes, but does not eliminate, the extent of these postirradiation conversions of oxymyoglobin to the ferri and ferryl derivatives. These observations are interpreted in terms of the scavenging of the 2-methyl-2-hydroxypropyl radicals by O 2 to generate their peroxy analogs, which causes a displacement of the equilibrium between oxy- and ferromyoglobin. The peroxy radicals decay to produce H 2O 2, an organic peroxide, and other products. These peroxides subsequently react with ferromyoglobin to produce the ferryl form; the rate of the reaction increases with decreasing [O 2] as [ferromyoglobin] increases. This reaction is sufficiently fast in deaerated solution that substantial conversion of ferromyoglobin to ferrylmyoglobin occurs during the time of irradiation. The formation of the ferryl derivative in the presence of unconverted ferromyoglobin drives a concurrent synproportion reaction which produces ferrimyoglobin. Overall, no direct interaction of 2-methyl-2-hydroxypropyl radicals, nor their peroxy analogs, with myoglobin is indicated; all reactivity is accountable by the peroxide products of these radicals.

The synproportionation reaction between the oxy and ferryl derivatives of myoglobin

The reaction between the oxy and ferryl derivatives of myoglobin to produce ferrimyoglobin has been studied in detail as functions of reactant concentrations, pH, ionic strength, [O 2], and temperature. The formation of ferrimyoglobin is shown to derive predominantly from a synproportionation of the oxy and ferryl derivatives rather than from a combination of the autoredox processes of each reactant. Details of the concurrent autoreduction of ferrylmyoglobin have also been examined. The results suggest that the active sites involved in the reactive encounter of the oxy and ferryl derivatives are of the same charge type and are effectively multiply charged. The acidic form of ferrylmyoglobin is at least an order of magnitude faster in reacting with MbO 2 than is its basic analog. The deoxy form of reduced myoglobin is marginally more reactive in the synproportionation process than the oxy analog.

Haem-apoprotein interactions detected by resonance Raman scattering in Mb- and Hb-derivates lacking the saltbridge His146 beta-Asp94 beta.

The dispersion of the depolarization ratio of oxidation- and spin-marker lines of sperm whale myoglobin derivatives (oxyMb, deoxyMb, ferric Mb-CN) and of ferric Hb-CN have been measured for different pH-values in the acid and alkaline region. No pH-dependence in the region above pH = 6.5 has been found. Below pH = 6.5, however, a significant pH-dependence of the oxyMb-oxidation marker line at 1,375 cm-1 exists. Additionally, a weak pH-dependence of the corresponding 1,355 cm-1 line of the deoxymyoglobin spectrum is observed. This effect can be explained assuming a titration of distal histidine, inducing a rupture of the ligand-imidazole H-bond in the case of oxymyoglobin. The pH-independent depolarization ratio disperson above pH = 6.5 in all systems investigated is explained by the lack of the haemoglobin saltbridge between His(HC3) beta and Asp(FG5) beta, which is essential for the cooperativity in the haemoglobin system.

Comparison of hydrogen–deuterium exchange in myoglobin derivatives reveals localized conformational stable regions

Comparison of hydrogen–deuterium exchange in myoglobin derivatives reveals localized conformational stable regions

Interaction of radiation-generated radicals with myoglobin in aqueous solution—II: Analysis of product yields for hydroxyl radicals with oxymyoglobin under deaerated conditions

Gamma-irradiation of N 2O-saturated aqueous solutions containing predominantly oxymyoglobin (with small amounts of ferrimyoglobin and deoxymyoglobin) leads to the near-quantitative conversion of the oxy derivative to the deoxy, ferri, and ferri-peroxide (ferryl) forms of myoglobin. All of the ferrimyoglobin-peroxide production is accountable by H 2O 2 involvement, indicating ·OH/H· participation in the formation of the other product derivatives. A methodology for compositional analysis of the component myoglobin derivatives in this complex system is developed.

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O 2-complexes of myoglobin and hemoglobin. Over all the spectral region (450–800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d → d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe- N ϵ , (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.

Experimental and Calculated Magnetic Circular Dichroism Spectra of Iron(II) Low Spin Hemoglobin and Myoglobin with CO, NO, and O2

Abstract The ratios of the apparent Faraday A terms to the dipole strengths (A′⁄D) were obtained from the experimental magnetic circular dichroism (MCD) data for the iron(II) low spin derivatives of human hemoglobin and its α and β chains, and myoglobins (whale and bovine). The observed MCD peak height and trough depth exhibit the decreasing order of CO>O2>NO in the Soret band and O2>CO>NO for the Q band. The moment analysis also showed the decreasing order of the A′⁄D values of CO>O2>NO in the Soret bands for all hemoglobin and myoglobin derivatives experimentally studied. The molecular orbital calculation of the porphyrin π electron system based on the PPP CI approximation method demonstrated that the calculated spectra for the pyrrole nitrogen core-charge of 1.5 showed characteristics for the CO complex of hemoglobin and myoglobin, and that the NO and O2 complexes could be simulated by the calculations for the core charge values smaller than 1.5 (i.e. 1.4–1.3). These results suggest that the change in the porphyrin π electronic state which is caused by the electronic interaction between the diatomic molecule such as CO, NO, or O2 and the heme iron can be explained by the change in the σ core charge in the pyrrole nitrogen possibly through changes in the electronic states of the heme iron.

Evidence for protein self-association induced by excluded volume Myoglobin in the presence of globular proteins

The fluorescence polarization of fluorescent derivatives of hemoglobin and myoglobin was measured as a function of the concentration of added polymers (PEG-6 000, PEG-20 000) and globular proteins (lysozyme, ribonuclease A, β-lactoglobulin). The results indicated that the effective size and shape of 1-anilino-9-naphthalene sulfonate myoglobin are unaltered in the presence of up to 25 g/dl poly(ethylene glycol), whereas they are significantly altered in the presence of comparable concentrations of other proteins. The results are consistent with the hypothesis that in the presence of high concentrations of added protein, 1-anilino-9-naphthalene sulfonate myoglobin self-associates to form a dimer similar in size and shape to 1-anilino-9-naphthalene sulfonate hemoglobin.

Formation of a steady state in the radiolysis of ferrimyoglobin in aqueous solution

The interaction of radiation-generated · OH radicals with ferrimyoglobin in deaerated aqueous solution at neutral pH has been quantitatively studied. Changes in the visible absorption spectrum have been analyzed on the basis of composition changes of the ferri, deoxy, and ferriperoxide forms of the metalloprotein. A postirradiation thermal process must be considered in order to evaluate the radical-induced composition changes. Initially, ·OH induces reduction of ferrimyoglobin to the deoxy form with a G value (molecular yield/100 eV of absorbed energy) in the zero-dose limit of 1.4 (±0.2). Radiation-generated H 2O 2 reacts with the ferrimyoglobin substrate to produce ferrimyoglobin peroxide with a G value of 0.7 (±0.1) in the zero-dose limit. At doses of >1 krad μ m −1 of myoglobin present, the composition of the three myoglobin derivatives reaches a radiolysis steady state. In this moderate-dose plateau region, this composition is 44% ferri, 18% deoxy, and 38% ferri peroxide. The · OH-induced hemoprotein radicals that do not initiate 1-eq redox conversions undergo reactions that generate dimer and other globin-modified material.

Kinetic studies of spin interconversion in ferric mixed-spin derivatives of myoglobin.

AbstractKinetic studies of spin interconversion in various derivatives of metmyoglobin such as the fluoride, aquo, hydroxide, azide, imidazole, and cyanide were performed by the coaxial‐cable temperature‐jump method. For all these derivatives, except fluoride and aquomyoglobin, a single relaxation was observed around 3 μsec. The rate constants and activation parameters for the spin interconversion were estimated and are discussed in comparison with those reported for the reaction of synthetic iron complex. Other hemoproteins such as cytochrome c and human hemoglobin were also examined, and the results were compared with those for myoglobin. The effect of buffer solution is also discussed.

The heme environment of leghemoglobins Absorption and circular dichroism spectra of artificial leghemoglobins and myoglobins

Artificial leghemoglobins were reconstituted from apoleghemoglobin and meso-, deutero- and diacetyldeuteroheme. Absorption and circular dichroism spectra of their high-spin and low-spin derivatives in the ferrous and ferric forms were recorded in the ultraviolet and visible wavelength regions. The substitution of the 2,4-side-chains of heme induced changes in the optical activity, reflecting alterations in the heme environment. The effect on the conformation of aromatic amino acid residues around heme obviously correlates with the sixth axial ligand and the spin state of iron. Absorption and CD spectra of the aquoferric derivatives of artificial myoglobins were recorded in comparison. Strongly electron-withdrawing acetyl side-chains at the 2,4-positions of diacetyldeuteroheme caused a change in the absorption spectra of aquoferric leghemoglobin and myoglobin towards low spin. On the basis of the spectra it was suggested that the displacement of the ferric iron from the pyrrole plane in leghemoglobin derivatives would be smaller than in the corresponding myoglobin derivatives.

Structural significance of the amino-terminal residues of sperm whale myoglobin

Following the development of a nondestructive synthetic procedure for rapid production of des-Val1-myoglobin in large quantities, the synthesis of a series of myoglobin derivatives varying in structure and charge in the NH2-terminal region was accomplished. In comparison to the untreated myoglobin, the des-Val1-myoglobin was found to possess at low pH a decreased stability and an increased net positive charge in the pH range 5.5-8.5. While the elevated net positive charge was no longer apparent after removal of the second residue, the instability of the molecule was found to be sharply increased. Substitutions of the first residue, directed toward elucidating its structural importance, included glutamic acid, lysine, and glycine. Addition of any of the three amino acids to the des-Val1-myoglobin was found to restore much of the acid stability, with the [Gly1]myoglobin appearing nearly identical with the native molecule. All three semisynthetic myoglobins showed potentiometric titration curves characteristic of their respective, substituted residue. Carbamylation of the NH2 terminal of myoglobin and des-Val1-myoglobin yielded two nearly identical molecules in terms of all physical properties examined. Consequently, it was concluded that the first residue primarily serves the function of maintaining the positively charged NH2 terminus a certain distance away from the beginning of the A helix and from the charge pair interaction of Lys-133 with Glu-6. In addition, through physical measurements of the des-Val1,Leu2-myoglobin prior and subsequent to carbamylation of the NH2 terminus, it was apparent that the stabilization conferred on the des-Val1-myoglobin by the second residue was dependent to a large degree upon the hydrophobic interactions of its side chain.

Soret circular dichroism of cobalt-substituted myoglobin and hemoglobin derivatives

Circular dichroic spectra in the Soret region were obtained for the following cobalt-substituted hemoproteins: CoMb ‡ ‡ Abbreviations used: Mb, myoglobin; Hb, hemoglobin; FeHbO 2, native oxyhemoglobin; CoHbO 2, cobalto-oxyhemoglobin; FeHb, native deoxyhemoglobin; CoHb, cobaltodeoxyhemoglobin; FeHb +, native ferrihemoglobin; CoHb +, cobaltihemoglobin; CoHbNO, nitrosylcobaltohemoglobin; FeHbNO, native nitrosylhemoglobin (similar abbreviations were used for myoglobin derivatives); c.d., circular dichroism; IHP, inositol hexaphosphate. , CoMbO 2, CoMbNO, CoMb +, CoHb, CoHbO 2, CoHbNO and CoHb + and compared with the corresponding spectra of the native species to delineate the sensitivity of Soret circular dichroism to ligation, quaternary structures, metal ion substitution and its magnetic moment. Soret rotational strengths, R, were calculated, and dissymmetry ratios were used to reveal hidden transitions. The results indicate that Soret circular dichroism is sensitive to the metal ion, its oxidation state, ligation and local environment but neither to quaternary structural changes as proposed by Ferrone & Topp (1975), nor to the magnetic moment of the metal ion as suggested by Li & Johnson (1969).

Assessment of relative content of myoglobin, oxymyoglobin and metmyoglobin at the surface of beef

A method of calculating the relative content of myoglobin, metmyoglobin and oxymyoglobin at the surface of beef is described. It is based on measurements of reflex attenuance of incident light at the isobestic points 572, 525, 473 and 730 nm. The latter value corresponds to the achromatic attenuance of light at the meat surface and can be used as an objective measure of its lightness. It was shown that the colour image of the meat surface, and the relative amounts of these three myoglobin derivatives, are influenced by the opacity of the surface layer. A variability in the light diffusing properties of the meat surface may be considered to be the primary factor in creating differences of colour perception of meat derived from animals of the same species and of the same approximate age.

Time-resolved resonance raman spectroscopy of hemoglobin derivatives: heme structure changes in 7 nanoseconds.

Resonance Raman spectra of oxyhemoglobin, deoxyhemoglobin, carboxyhemoglobin, and the corresponding myoglobin derivatives have been obtained with 7-nanosecond laser pulses at 531.8 nanometers. The results suggest that no transient constraint of the heme group by the globin structure occurs on this time scale, and thus establish a temporal sequence for the early events that may participate in the stereochemical trigger mechanism of hemoglobin cooperativity.

Analysis of myoglobin derivatives in meat or fish samples using absorption spectrophotometry.

Analysis of myoglobin derivatives in meat or fish samples using absorption spectrophotometry.