Reduction of ferrylmyoglobin to metmyoglobin by quinonoid compounds

Several quinoid compounds mediated the reduction of ferrylmyoglobin (MbIV) to metmyoglobin (MbIII). The efficiency of the MbIV reduction to MbIII was accomplished by the quinones in the following order: p-benzoquinone greater than 1,4-naphthoquinone greater than 2-OH-1,4-naphthoquinone greater than 2,3-epoxy-1,4-naphthoquinone. The quinone-mediated reduction of MbIV to MbIII had the following characteristics: (a) it was stoichiometrically--rather than catalytically--related to the number of cycles of the MbIV----MbIII transition involving the reduction of H2O2. (b) It proceeded with similar efficiencies under aerobic and anaerobic conditions. (c) It did not require the free radical form of MbIV(.MbIV), thus excluding a two-electron oxidation of the quinone. (d) the nucleophilic addition of--NH2 groups of the apoprotein on the quinone seemed not to be involved through an alternative pathway in the reduction of MbIV, especially since 2-OH-1,4-naphthoquinone, a compound which cannot undergo nucleophilic addition, also facilitated the reduction of the ferryl compound. (e) No two-electron oxidation products of the unsubstituted quinones, such as quinone epoxides, were detected in the spent reaction mixture analyzed by HPLC with electrochemical detection. On the basis of these observations, it is suggested that the reduction of MbIV to MbIII by the above quinonoid compounds is a one-electron transfer process, with electron abstraction being probably accomplished at some site in the benzo ring of the quinone.     

Conformational properties of native sperm whale apomyoglobin in solution.

Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JalphaH-NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X-PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H-helix. The EF-F-FG segment remains uncharacterized.     

Inhibition of human sperm acrosin by synthetic agents.

Twenty-two synthetic proteinase inhibitors were tested for their inhibitory properties towards human acrosin. p-Nitrophenyl-p1-guanidino benzoate (NPGB) was the most effective (K1 value of 1-5 X 10(-8) M), producing a non-competitive type of inhibition in contrast to all other inhibitors which showed a competitive type of inhibition. The Michaelis constant for human acrosin on BAEE at pH 8-1 was calculated to be 4-25 X 10(-5) M.     

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.     

Reduction of ferrylmyoglobin by the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) in aqueous solution and during freezing

The hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found to be reduced in a second-order reaction by N-tert-butyl-alpha-phenylnitrone (PBN, often used as a spin trap). In acidic aqueous solution at ambient temperature, the reduction is relatively slow (deltaH++ = 65+/-2kJ x mol(-1) and deltaS++ = -54+/-7 J x mol(-1). K(-1) for pH = 5.6), but phase transitions during freezing of the buffered solutions accelerates the reaction between ferrylmyoglobin and PBN. In these heterogenous systems at low temperature (but not when ice-formation was inhibited by glycerol), a PBN-derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl radical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and from microwave power saturation behavior concluded not to be located in the heme-cleft of the protein. The acceleration of the reaction is most likely caused by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing.     

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)     

Oxidation of sperm whale oxymyoglobin, catalyzed by ferrocyanide ions: kinetics and mechanisms

Specific catalytic oxidation of sperm whale oxymyoglobin by small amounts of potassium ferri- and ferrocyanide, from 1 to 20% in relation to the protein concentration, was studied. The mechanism of catalysis was shown to involve specific binding of the ferrocyanide anion to the protein. The influence of pH and ionic strength of the medium, the [Fe(CN)6]4- concentration and of chemical modification of Mb histidines by bromoacetate, as well as the effect of the Mb complexing with redox-inactive zinc ion on the rate of reaction was examined. The zinc ion forms a stable complex with His 119(GH1) on the Mb surface at the equimolar Zn2+ concentration. The kinetic scheme of the reaction was analyzed, and the equilibrium and kinetic parameters were obtained. It was first shown that the strong oxidant such as potassium ferricyanide is able to react with the same protein by two distinct mechanisms: (i) a simple outer sphere electron transfer over the heme edge and (ii) electron transfer after the specific binding of [Fe(CN)6]4- to oxyMb in the His 119(GH1) region, thus catalyzing the protein oxidation.     

Characterization of the reaction of methyl acetimidate with sperm whale myoglobin.

The effects of pH, acetimidate concentration, temperature, and reaction time of methyl acetimidate with sperm whale myoglobulin have been assessed. Reaction at pH 9.8 and 15 degrees C for 30 min with a sixfold excess of methyl acetimidate relative to each amino group yielded six acetimidomyoglobin derivatives which were separated and purified. Reaction with tetrahydrophthalic anhydride revealed the number of amino groups that remained unreacted in each separated component and made possible further subractionation. Modification at the NH2 terminus was quantitated by automated stepwise Edman degradation. The acetimidyl and tetrahydrophthalyl groups, were readily removable. The potentiometric titration of three of the completely deprotected components showed identity with the parent untreated sperm whale myoglobin. The first of two major products was acetimidated at all 19 epsilon-amino groups but not at the NH2 terminus. The second major product bore a blocked NH2 terminus but retained one unmodified epsilon-amino group, identified after modification by trinitrobenzenesulfonate as lysine residue 77. Of the minor components, one was identified as completely acetimidated at all 20 amino groups. The other three minor components appeared to contain irreversible by-products.     

Binding of alkylisocyanides with soybean leghemoglobin. Comparisons with sperm whale myoglobin

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are slower. For a given protein, the dissociation rate constants are not much different when different isocyanides are used (except for methyl), whereas the association rates show complex behavior in relation with the alkyl chain length; singular differences are observed between leghemoglobin and sperm whale myoglobin in this regard. For myoglobin, the binding rate constants decrease from methyl to n-propyl, but remain approximately the same when the ligand carries a still longer alkyl chain. In contrast, for leghemoglobin, although the rate constants decrease from methyl to n-propyl, they show a progressive and important rise with longer alkyl substituents: n-butyl and n-pentyl.     

Effect of reducing agents and uncouplers on the electrical potential generated by mitochondrial ATPase activity

Beef heart submitochondrial particles bound to phospholipids impregnated filters generated an electrical potential upon the addition of ATP. The magnitude of the electrical potential reached depended on the phospholipid mixture composition used for filter impregnation, phosphatidylethanolamine being the active component for the electrical potential generation. Uncoupler FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone) inhibited the transmembrane electrical potential generation by diminishing the electrical resistance of the system as a result of its protonophoric action. However, uncouplers 2, 4-dinitrophenol and dicoumarol did not provoke large modifications of the electrical resistance under the conditions of pH and concentration used, and their action varied with the time elapsed after the submitochondrial particles purification, favouring the idea of the uncoupler interaction with a specific site on the membrane. Addition of sodium dithionite resulted in a higher plateau value for the electrical potential consistent with the promoted increase in ATPase activity. The effect of this agent was reversed by the 2,6-dichlorophenol-indophenol added at equivalent concentrations.     

Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions

The electrostatic free energy contribution to the stability of sperm whale ferrimyoglobin was evaluated according to the static accessibility modified Tanford-Kirkwood model. The electrostatic free energy contribution of each distinct structural element was divided into one term arising from interactions between it and other elements (interelemental) and another from interactions within the particular element itself (intraelemental). At pH 7 the majority of the terms were found to be stabilizing. The interelemental terms are the dominant ones for most structural elements. The small interelemental terms of the C and D helices are compensated by large intraelemental interactions which stabilize these short helices. Perturbations in pH can be accommodated by the structural elements through a redistribution of stabilizing and destabilizing interactions. The electrostatic potentials calculated at the surface of the protein indicate that the internal compensation of local potentials achieved during folding results in a generally neutral protein-solvent interface save for two distinct areas of nonzero potential. The accessibility of each charged atom to solvent was analyzed in terms of the surface area lost to charged, polar and nonpolar atoms separately. The net solvent accessibility lost parallels closely that lost to nonpolar atoms alone, indicating a specific role for nonpolar atoms in defining dielectric shielding of charged atoms, aside from their participation in the well-known hydrophobic interactions.     

Deuterium nuclear magnetic resonance of deuterium-labeled diacetyldeuterohemin incorporated into sperm whale myoglobin.

The heme derivative 2,4-diacetyldeuterohemin deuterated in the methyl groups of the acetyl moieties was reconstituted with sperm whale apomyoglobin and the two labeled methyl groups in the protein environment were observed by deuterium nuclear magnetic resonance spectroscopy. The results were compared to the free hemin form as the dimethyl ester in chloroform and in a pyridine-water mixture, as well as in the zinc complex form. Under most conditions the two methyl resonances overlie each other to a large degree. Resonance width at half-height is of the order of 25 Hz for the protein and approximately one-third as much for the free hemin at 16 degrees and is little affected by conversion to paramagnetic derivatives. Chemical shifts for the oxy- and carbonmonoxymyoglobins are very similar. In cyanoferrimyoglobin a positive pseudo-contact contribution of 3.04 ppm was computed to explain a relative upfield shift offset in part by a small negative contact shift contribution. The cyanoferrimyoglobin resonance was sensitive to the presence of phosphate buffer as well as to cyclopropane. The aquoferrimyoglobin form shows distinct resonances for the two methyl groups, with the downfield resonance considerably broadened. The expected effects of temperature on chemical shift were observed, the paramagnetic derivatives showing an effect and carbonmonoxymyoglobin showing none. The relaxation behavior was gauged from the line widths and from measurements of spin-lattice relaxation time, T1. The effective rotational correlation time is of the order of 50 ps for the liganded myoglobin forms. The temperature dependence of the line widths may imply an increased retational freedom with increasing temperature. The broadening observed in the aquoferrimyoglobin case is indicative of restricted internal rotational motion of one of the methyl groups. The method is suitable for probing the more mobile structures in proteins and retains its value in the neighborhood of paramagnetic centers.     

Dielectric properties of hemoglobin and myoglobin. II. Dipole moment of sperm whale myoglobin

This paper is concerned with the molecular origin of the dipole moment of sperm whale myoglobin as it can be calculated from the dielectric dispersion at 1 Mcps on the basis of a mechanism of orientational polarization. It was possible to compare the dielectric increment of native myoglobin and its change during the reaction with bromo acetate with dipole moments calculated according to the known coordinates of the charged groups of the molecule. The agreement between the two shows that in myoglobin only the permanent dipole moment due to these charged groups is important, and that contributions from other possible sources remain within the limits of experimental error.     

Optical spectroscopic observation of a metastable form of sperm whale myoglobin generated by reconstitution

The optical spectrum of Sperm Whale myoglobin, which has been freshly reconstituted with iron protoporphyrin-IX, is shown to be different from that obtained from the native myoglobin, and from the reconstituted, incubated myoglobin (These last two have equivalent absorption spectra). The effect is immediately evident as a shift of about +1 nm in the Soret band during incubation of freshly reconstituted metMb. Difference spectroscopy can be used to deconvolute changes in optical spectra occurring during and after Mb reconstitution into two components. The initial phase reflects incorporation of hemin into the protein matrix; this is already known to produce two forms, differing by relative hemin orientation. The rate of the second process follows the known pH dependence of iron protoporphyrin-IX reorientation. Presence of the second process indicates that the absorption spectrum of each of the two hemin-insertion Mb forms is unique, so interconversion between the two forms is monitored. Thus iron protoporphyrin-IX reorientation in proteins may be studied by visible spectroscopy.     

Effect of reducing agents on oxidation-reduction potential and the outgrowth of Clostridium botulinum type E spores.

Oxidation-reduction potential (Eh) levels were measured and standardized to pH (Eh7) for Trypticase soy broth containing various concentrations of reducing agents. Prereduced Trypticase soy broth with no added reducing agents exhibited a potential of -141 mV. Ascorbic acid at 0.2 to 0.005% and sodium thioglycolate at concentrations below 0.05% produced an Eh7 higher than the prereduced Trypticase soy broth containing no added reducing agents. The addition of cysteine hydrochloride,2-mercaptoethanol, and sodium formaldehyde sulfoxylate to prereduced Trypticase soy broth resulted in a reduction of Eh7 compared to the system without added reducing agents. The order of relative reducing intensity (from highest to lowest) for the reducing agents when comparing molar concentration was: sodium formaldehyde sulfoxylate,2-mercaptoethanol, cysteine hydrochloride, sodium thioglycolate, and ascorbic acid. Optimal growth of the test organism occurred at low Eh7 and low concentration of the reducing agents. A direct correlation existed between growth of the test organism and -Eh7 x -log concentration of the reducing agent.