The mechanism of oxymyoglobin oxidation catalyzed by ferrocyanide ions: chemically modified and mutant sperm whale myoglobins

A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.     

The oxidation of sperm whale, horse, and pig oxymyoglobins, catalyzed by ferrocyanide ions: kinetics and mechanism

The influence of pH, ionic strength of the solution, and [Fe(CN)6]4- concentration on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins, which is catalyzed by ferrocyanide ions, was studied. These myoglobins have homologous spatial structures and identical redox potentials but differ by the amount of His residues located on the protein surface. The effect of the MbO2 complexing with redox-inactive Zn2+ ion on the reaction rate was also examined. At the equimolar Zn2+ concentration, zinc ions form a stable complex with His119(GH1). It was found that the kinetic behavior of horse MbO2, which lacks His12(A10) substituted for by Gln, is fully analogous to one of sperm whale MbO2, while the oxidation of pig MbO2, three histidines of which, His12, His113(G14), and His116(G17), are replaced by Gln, is strongly inhibited. The mechanism of the catalysis was shown to involve specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) site, which is in accord with the large positive electrostatic potential of this site and the presence here of a cavity large enough to accommodate [Fe(CN)6]4-. The nearby His113 and His116 residiues, which are absent in pig Mb, also play a very important role in the catalysis, because their protonation (especially of the last residue) is most likely responsible for the week oxidation of bound [Fe(CN)6]4- by dissolved oxygen.     

Stability properties of sperm whale oxymyoglobin

Sperm whale oxymyoglobin was isolated directly from muscle and was examined for its stability properties over the wide range of pH 5–13 in 0.1 m buffer at 25 °C. The remarkable pH dependence for the autoxidation rate was analyzed using the kinetic equation derived in terms of nucleophilic displacement processes of O₂^? from oxymyoglobin by the entering water molecule or hydroxyl ion with the iron resulting in the ferric form. Most of the autoxidation reaction of the oxymyoglobin can be best explained by the proton-catalyzed processes involving the distal histidine as the catalytic residue. The kinetic equation could also be used as an interesting diagnostic probe into differences in the heme reactivity and the heme environment of different types of oxymyoglobin from other sources.     

Mechanism of oxidation of oxymyoglobin by copper ions: comparison of sperm whale, horse, and pig myoglobins

The influence of Cu²⁺ concentration, pH, and ionic strength of the solution as well as redox-inactive zinc ions on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. These myoglobins have homologous spatial structures and equal redox potentials but differ in the number of histidines located on the surface of the proteins. It was shown that oxy-Mb can be oxidized in the presence of Cu²⁺ through two distinct pathways depending on which histidine binds the reagent and how stable the complex is. A slow pH-dependent catalytic process is observed in the presence of equimolar Cu²⁺ concentration for sperm whale and horse oxymyoglobins. The curves of pH dependence in both cases are sigmoid with pK _eff corresponding to the ionization. The process is caused by the strong binding of Cu²⁺ to His113 and His116, an analogous His residue being absent in pig Mb. In contrast, rapid oxidation of 10-15% of pig oxy-Mb is observed under the same conditions (fast phase), which is not accompanied by catalysis because the reduced copper is apparently not reoxidized. The complexing of Cu²⁺ with His97 situated near the heme is probably responsible for the fast phase of the reaction. The affinity of His97 for Cu²⁺ must be significantly lower than those of the catalytic His residues since the fast phase does not contribute markedly to the rate of sperm whale and horse oxy-Mb oxidation. Increasing copper concentration does not produce a proportional growth in the oxidation rate of sperm whale and horse oxy-Mbs. Which Cu²⁺ binding sites of Mb make main contributions to the His reaction rate at different Cu²⁺/Mb ratios from 0.25 to 10 is discussed.     

Effect of artificial and natural phospholipid membranes on rate of sperm whale oxymyoglobin autooxidation

We were the first to show that MbO₂ deoxygenation in the cell occurs only upon interaction of myoglobin with mitochondrial membrane, which must be accompanied by changes in the heme cavity conformation of the protein and its affinity for the ligand. Under aerobic conditions, some changes in the equilibrium O₂ dissociation constant (K _dis) can be detected by changes of the rate of MbO₂ autooxidation, i.e. spontaneous turning it into metMb (k _ox), as far as a direct correlation between K _dis and k _ox is experimentally shown. In this work, we studied the effect on MbO₂ autooxidation rate of phospholipid liposomes from neutral soybean phosphatidylcholine (lecithin) and from negatively charged 1-palmitoyl-2-oleylphosphatidylglycerol (POPG) at various phospholipid/MbO₂ ratios from 25 : 1 to 100 : 1, and also the effect of rat liver mitochondria at concentration of 1 and 2 mg/ml mitochondrial protein (at 22 and 37°C). In all cases, k _ox was found to increase due to interaction of the protein with phospholipid membranes. The effect of negatively charged liposomes from POPG on kox is significantly greater than that of neutral lecithin liposomes. At the POPG/MbO₂ molar ratio of 25 : 1, MbO₂ autooxidation rate is almost 25-fold increased compared to the control, whereas in the presence of 50-fold molar excess of lecithin, k _ox is only ～10 times higher (10 mM buffer, pH 7.2, 22°C). With the same phospholipid/MbO₂ ratio of 100: 1, k _ox is 7 times higher for the POPG than for lecithin liposomes. In the presence of mitochondria inhibited by antimycin A, k _ox grows proportionally to their concentration (about 10-fold per 1 mg/ml of mitochondrial protein), and practically does not change after adding superoxide dismutase in the reaction mixture. The k _ox value decreases markedly at high ionic strength, thus suggesting an important role of coulombic electrostatics in the myoglobin-mitochondrial interaction. The increase in the autooxidation rate of MbO₂ (and hence its K _dis) due to the interaction with phospholipid membranes points to decreasing affinity of myoglobin for oxygen, which facilitates O₂ detachment from MbO2 at physiological p ₀₂ values.     

New transient species of sperm whale myoglobin in photodissociation of dioxygen from oxymyoglobin

We carried out the flash photolysis of oxy complexes of sperm whale myoglobin, cobalt-substituted sperm whale myoglobin, and Aplysia myoglobin. When the optical absorption spectral changes associated with the O2 rebinding were monitored on the nanosecond to millisecond time scale, we found that the transient spectra of the O2 photoproduct of sperm whale myoglobin were significantly different from the static spectra of deoxy form. This was sharply contrasted with the observations that the spectra of the CO photoproduct of sperm whale myoglobin and of the O2 photoproducts of cobalt-substituted sperm whale myoglobin and Aplysia myoglobin are identical to the corresponding spectra of their deoxy forms. These results led us to suggest the presence of a fairly stable transient species in the O2 photodissociation from the oxy complex of sperm whale myoglobin, which has a protein structure different from the deoxy form. We denoted the O2 photo-product to be Mb*. In the time-resolved resonance Raman measurements, the nu Fe-His mode of Mb* gave the same value as that of the deoxy form, indicating that the difference in the optical absorption spectra is possibly due to the structural difference at the heme distal side rather than those of the proximal side. The structure of Mb* is discussed in relation to the dynamic motion of myoglobin in the O2 entry to or exit from the heme pocket. Comparing the structural characteristics of several myoglobins employed, we suggested that the formation of Mb* relates to the following two factors: a hydrogen bonding of O2 with the distal histidine, and the movement of iron upon the ligation of O2.     

Preferential uptake of ammonium ions by zinc ferrocyanide

The concentration of ammonia from dilute aqueous solution could have facilitated many prebiotic reactions. This may be especially true if this concentration involves incorporation into an organized medium. We have shown that (unlike iron(III) ferrocy anide) zinc ferrocyanide, Zn₂ Fe (CN)₆·xH₂O, preferentially takes up ammonium ions from 0.01 M NH₄Cl to give the known material Zn₃(NH₄)₂ [Fe(CN)₆]₂·xH₂O, even in the presence of 0.01M KC1. KC1 alone gave Zn₃K₂[Fe(CN)₆]₂·xH₂O. Products were characterized by elemental (CHN) analysis and powder X-ray diffraction (XRD). We attribute the remarkable specificity for the ammonium ion to the open framework of the product, which offers enough space for hydrogen-bonded ammonium ions, and infer that other inorganic materials with internal spaces rich in water may show a similar preference.     

Myoglobin and mitochondria: kinetics of oxymyoglobin deoxygenation in mitochondria suspension

The kinetics of whale MbO2 deoxygenation was studied spectrophotometrically in the presence of breathing rat mitochondria under conditions when mitochondria were separated from the protein solution by a semipermeable film capable to transfer only low-molecular-weight compounds and directly in the solution of MbO2 with mitochondria (incubation medium: 15-35 mM succinate, 150 mM sucrose, 100 mM KCl, 0.5 mM EGTA, 5 mM KH2PO4, 10 mM MOPS, pH 7.4). It was shown that the splitting of O2 from MbO2 at physiological pO2 is possible only if it directly contacts mitohondria. The deoxygenation rate does not depend on the protein concentration (zero order on [MbO2] as opposite to the first order reaction in the absence of mitochondria) and completely coincides with the rate of oxygen consumption by mitochondria under the same conditions, as indicated by the polarographic data. The dependence of the MbO2 deoxygenation rate on the concentration of mitochondria and the protein, and on the total charge of the MbO2 molecule was studied using horse MbO2 (pI 7.1), sperm whale MbO2 (pI 8.3), its zinc complex, Zn-MbO2 (pI > 8.3), and the sperm whale MbO2 derivative carboxymethylated at His residues, CM-MbO2 (pI 5.2). The mechanism of MbO2 deoxygenation in the cell obviously actuates its interplay with the mitochondrial membrane. As a result, the affinity of Mb to oxygen decreases several times, which corresponds to a shift of the Mb dissociation curve to higher pO2 values.     

Oxidation of oxymyoglobin by nitric oxide through dissociation from cobalt nitrosyls

Kinetic evaluation of the oxidation of oxymyoglobin (MbO₂) to metmyoglobin (Mb⁺) by bis(dimethylglyoximato)cobalt nitrosyl [Co(NO)(DMGH)₂] has established that the mechanism of this transformation involves initial dissociation of nitric oxide from Co(NO)(DMGH)₂, followed by direct oxidation of MbO₂ by nitric oxide. Nitrate formation accompanies the production of Mb⁺ and is proposed to arise from isomerization of the initially formed peroxynitrite ion. By comparative kinetic determinations with nitrosyl transfer from the cobalt nitrosyl reagent to deoxyhemoglobin, the rate constant for oxidation of MbO₂ by nitric oxide is calculated to be 31 X 10⁶ M^?1sec^?1 at 10.0°C in phosphate-buffered media at pH 7.0. Bis(dimethylglyoximato)cobalt(II), the cobalt complex formed by nitric oxide dissociation from Co(NO)(DMGH)₂, is an effective trap for dioxygen liberated from MbO₂. The resulting μ-peroxo- or μ-superoxo-dicobaloxime(III) oxidizes deoxymyoglobin to metmyoglobin at a rate that is competitive with oxidation induced by Co(NO)(DMGH)₂.     

Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of ferryl intermediate

Hydrogen peroxide, one of the potent oxidants in muscle tissues, can induce very rapid oxidation of oxymyoglobin (MbO2) to metmyoglobin (metMb) with an apparent rate constant of 7.5 X 10(4) h-1 M-1 (i.e., 20.8 s-1 M-1) over the wide pH range of 5.5-10.2 in 0.1 M buffer at 25 degrees C. Its molecular mechanism, however, is quite different from that of the autoxidation of MbO2 to metMb. Kinetic analysis has revealed that the hydrogen peroxide oxidation proceeds through the formation of ferryl-Mb(IV) from deoxy-Mb(II), which is in equilibrium with MbO2, by a two-equivalent oxidation with H2O2. Once the ferryl species is formed, it reacts rapidly with another deoxy-Mb(II) in a bimolecular fashion so as to yield 2 mol of metMb(III). Under physiological conditions, the rate-determining step was the oxidation of the deoxy species by H2O2, its rate constant being estimated to be on the order of 3.6 X 10(3) s-1 M-1 at 25 degrees C. These findings leads us to the view that a good supply of dioxygen provides rather an important defense against the oxidation of myoglobin with hydrogen peroxide in cardiac and skeletal muscle tissues.     

The mechanism of oxymyoglobin oxidation by copper ions and complexes: myoglobins carboxymethylated and carboxyamidated at histidine residues

The oxidation of sperm whale oxymyoglobin (MbO2) and its chemically modified derivatives alkylated at solvent-accessible histidines by sodium bromoacetate (CM-MbO2) and iodoacetamide (CA-MbO2) in the presence of ions and glycine complexes of copper, Cu2+, Cu(Gly)+, and Cu(Gly)2, has been studied. The influence of the reagent concentration, pH, and ionic strenth of medium, and also of competitive redox-inactive zinc ions on the reaction was investigated. The localization of Cu(Gly)2 in native sperm whale met-Mb and CM-met-Mb was examined by the high-resolution NMR method. The data obtained confirm that the linkage of copper compounds to surface histidines (all of them are away from the heme, at a distance of 1.8-2.7 nm) has only a minor (no more than 35%) contribution to the overall reaction rate, in particular under conditions of a large, more than 8-10-fold, data, excess of the reagent. The noticeable contribution of His116(113), His48, and His81, which according to NMR are localized on the protein surface and have the greatest affinity to copper, is revealed only at small concentrations of copper, a less than 5-fold excess relative to the protein. This is supported by the sigmoidal pH-dependence curve with the transition pK 6.5 at the equimolar copper concentration. The main contribution to the rate of the reaction studied should involve a linkage of copper to internal histidines, His97(FG4), which is 0.66 nm apart from the heme, and to distal His64(E7). Both are hydrogen bonded, the first with carboxyl group of one of the heme propionates, and the second with liganded O2, have a much lower affinity to copper than surface histidines, and are inaccessible to the modification.     

Oxidation of 6-hydroxydopamine catalyzed by tyrosinase

• 1.1. The oxidation of 3,4-dihydroxyphenylethylamine (dopamine) by O₂ catalyzed by tyrosinase yields 4-(2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaminequinone-H⁺, which evolves non-enzymatically through two branches or sequences of reactions, whose respective operations are determined by the pH of the medium.
• 2.2. The cyclization branch of o-dopaminequinone-H⁺ takes place in the entire range of pH and is the only significant branch at pH ⩾ 6.
• 3.3. The hydroxylation branch of o-dopaminequinone-H⁺ only operates significantly at pH < 6, and involves the accumulation of 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine), identified by high performance liquid chromatography (HPLC).
• 4.4. 6-hydroxydopamine is also a substrate of tyrosinase. The identification and evolution of the oxidation products of 6-hydroxydopamine has been carried out by spectrophotometry and HPLC assays.

     

Primary folding dynamics of sperm whale apomyoglobin: core formation

The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH(*) 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of approximately 26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 micro s for a 7 degrees jump to 52 degrees C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 micro s and 56 micro s for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)