Spectral studies of magnesium porphyrin--apomyoglobin and apohemoglobin complexes

Complexes of magnesium protoporphyrin and magnesium mesoporphyrin with apomyoglobin and apohemoglobin have been prepared and studied by electronic, circular dichroism, and optical rotatory dispersion spectroscopy. The myoglobin complexes show prominent splittings and red shifts of the visible absorption bands, with respect to those for the hemoglobin analogs. Comparisons are made with other heme protein systems that display similar spectral features. Different reasons for the observations are considered, including multiple conformer formation, polarity effects of the protein environment, and the formation of six-coordinate magnesium species through H2O coordination for the Mb complexes (the latter explanation being favored).     

A comparative spectroscopic and calorimetric investigation of the interaction of amsacrine with heme proteins,hemoglobin and myoglobin

The binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21?±?.05) × 10⁵ M^?1, while that to myoglobin was three times higher (3.59?±?.15) × 10⁵ M^?1. The temperature-dependent fluorescence study confirmed the formation of ground-state complexes with both the proteins. The stronger binding to myoglobin was confirmed from both spectroscopic and calorimetric studies. The binding was exothermic in both cases at the three temperatures studied, and was favored by both enthalpy and entropy changes. Circular dichroism results, three-dimensional (3D) and synchronous fluorescence studies confirmed that the binding of amsacrine significantly changed the secondary structure of hemoglobin, while the change in the secondary structure of myoglobin was much less. New insights, in terms of structural and energetic aspects of the interaction of amsacrine with the heme proteins, presented here may help in understanding the structure-activity relationship, therapeutic efficacy, and drug design aspects of acridines.     

Low temperature photodissociation studies of ferrous hemoglobin and myoglobin complexes by Mössbauer spectroscopy

⁵⁷Fe-enriched complexes of hemoglobin and myoglobin with CO and O₂ were photodissociated at 4.2°K, and the resulting spectra were compared with those of the deoxy forms. Differences in both quadrupole splitting and isomer shift were noted for each protein, the photoproducts having smaller isomer shift and larger quadrupole splitting than the deoxy forms. The photoproducts of HbCO and HbCO₂ had narrow absorption lines, indicating a well-defined iron environment. The corresponding myoglobin species had broader absorption lines, as did both deoxy forms. The weak absorption lines of photodissociated NO complexes appeared to be wide, possibly indicating magnetic interaction with the unpaired electron of the nearby NO.     

Structure-Function Relationships in Unusual Nonvertebrate Globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear.

In this review, special emphasis is placed on the stability properties of the heme-bound O₂. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O₂ to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO₂ or HbO₂ are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

Low temperature photodissociation studies of ferrous hemoglobin and myoglobin complexes by M?ssbauer spectroscopy.

57Fe-enriched complexes of hemoglobin and myoglobin with CO and O2 were photodissociated at 4.2 degrees K, and the resulting spectra were compared with those of the deoxy forms. Differences in both quadrupole splitting and isomer shift were noted for each protein, the photoproducts having smaller isomer shift and larger quadrupole splitting than the deoxy forms. The photoproducts of HbCO and HbO2 had narrow absorption lines, indicating a well-defined iron environment. The corresponding myoglobin species had broader absorption lines, as did both deoxy forms. The weak absorption lines of photodissociated NO complexes appeared to be wide, possibly indicating magnetic interaction with the unpaired electron of the nearby NO.     

CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin.

The effects of pH upon infrared spectra [CO stretching frequency (vco) region] and visible spectra of the CO complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A are reported. The vco for leghemoglobin--CO complexes was 1947.5 cm-1 at neutral pH. At acid pH myoglobin-- and hemoglobin--CO complexes developed vco bands at 1966--1968 cm-1, whereas leghemoglobin--CO complexes developed vco bands at approximately 1957 cm-1. All pKapp co values determined by pH-dependent variation of vco fell in the range 4.0--4.6. The pKapp co values determined from visible spectra were consistent with vco-determined values except for that of myoglobin--CO (visible pKapp co = 5.8). The pKapp co values in the 4.0--4.6 range appear to be pK values of the distal histidines, while the visible pKapp co of myoglobin--CO appears to be the pK of a group other than the distal and proximal histidines. The data are consistent with a model in which protonation of the distal histidine permits protein-free heme FeCO geometry in leghemoglobin--CO complexes but not in myoglobin-- or hemoglobin--CO complexes. Thus the heme pockets of leghemoglobins appear to be more flexible than the heme pockets of myoglobin and hemoglobin. The effects of pH upon visible spectra of the O2 complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A also are reported. pKapp o2 values of approximately 5.5 (leghemoglobins) and 4.4 (hemoglobin) are probably the pK values of the distal histidines. Comparisons of pKapp o2 values with pKapp co values indicate a more flexible heme pocket in leghemoglobins than in hemoglobin. The O2 complex of leghemoglobin c2 differed significantly from the O2 complexes of leghemoglobins a and c1 in visible spectra and titration behavior. These differences might be associated with the small structural differences in the region between the E and F helixes of leghemoglobins.     

Reassignment of residue 122 in the myoglobin from the killer whale,Orcinus orca

Summary The complete amino acid sequence of the major component myoglobin from killer whale,Orcinus orca, was determined by automated Edman degradation. In this study residue 122 was found to be glutamic acid instead of glutamine as was originally reported (Castillo et al. 1977). This reassignment affects the phylogenetic relationship of killer whale myoglobin with the myoglobins from other closely related cetacean species and also affects studies concerned with the physical parameters of the protein.This is the 114th paper in a series dealing with coordination complexes and catalytic properties of proteins and related substances. For the preceding paper see Neireiter et al. 1979. This work was supported by Public Health Service Research Grant HL-05556     

Reactive complexes in myoglobin and nitric oxide synthase

In this overview some of our crystallographic and spectroscopic studies on reactive complexes in myoglobin and nitric oxide synthase are summarised. Myoglobin and nitric oxide synthase are both haemoproteins with some similar reaction intermediates. For myoglobin we have studied different intermediates generated in the reaction with hydrogen peroxide by X-ray diffraction, single-crystal microspectrophotometry, electron paramagnetic resonance spectroscopy, Mössbauer spectroscopy, resonance Raman spectroscopy and quantum refinement. Several of these myoglobin states are quite susceptible to radiation-induced changes during crystallographic data collection, and we have observed a radiation-induced change of the ferric resting myoglobin to aqua ferrous myoglobin, of myoglobin compound II to a proposed intermediate H, and of myoglobin compound III to peroxy myoglobin. For the myoglobin compound II/ intermediate H we observe a single-bonded Fe^IV-O species, which is probably protonated. The long Fe-O bond seen in the crystal structure can be supported by the observation of a new ¹⁸O-sensitive resonance Raman mode at 687 cm⁻¹. For nitric oxide synthase we detected with cryobiochemical methods in electron paramagnetic resonance spectra the first biopterin radical serving as electron donor to the ferrous-oxy complex, and that biopterin serves as a proton donor as well, in addition we could observe formation of the Fe(NO) complex with a amino-pterin cofactor capable to form a reactive radical.     

Time Kinetics of Hemoglobin and Myoglobin Activation by Tetrachlorodecaoxide (TCDO)

In the presence of peroxidase, myoglobin or hemoglobin, Tetrachlorodecaoxide (TCDO) forms an active oxygen species which is similar to the product of the polymorphonuclear leucocyte (PMNL) myeloperoxidase reaction and the “Klebanoff Model” of phagocytosis, but it is also produced under anaerobic conditions. Randomly destructive species such as the free OH^- radical or singlet oxygen are not formed. The kinetics of the heme-dependent activation vary according to the heme type present. In comparison to myoglobin, blood shows a 2 h delay in the appearance of maximal activity. On the basis of known biochemical and clinical-physiological data, a hypothesis can be proposed to explain the reoxygenation observed in hypoxic tissue, induced by TCDO via this activated heme species. Under normal physiological conditions, vasodilation occurs via catalysis by xanthine oxidase or PMNL-dependent activation of fatty acids.     

Factors affecting the metal ion-hydroxamate interactions II: effect of the length of the connecting chain on the Fe(III), Mo(VI) and V(V) complexation of some new desferrioxamine B (DFB) model dihydroxamic acids

Three new dihydroxamic acids (HO(CH₃)NCO-(CH₂)₂-CO-NH-(CH₂)_x-CON(CH₃)OH where the x values are 4; 3 and 2, and the compounds are abbreviated as 2,4-DIHA, 2,3-DIHA and 2,2-DIHA), containing the peptide group in a certain position to one of the two functional groups and in different distances to the other one, were synthesized and their complexation with Fe(III), Mo(VI) and V(V) was studied by pH-potentiometric, spectrophotometric and in some cases by CV methods to evaluate the redox behaviour of the Fe(III) complexes and assess their potential biological activity as siderophore models. All these compounds are structural models for the natural siderophore, desferrioxamine B (DFB). The results were compared to those of the complexes of 2,5-DIHA having the same connecting chain structure and length as DFB has, and the effects of the length of the connecting chain on the co-ordination mode and on the stability of the complexes formed were evaluated.Very similar stability of the mono-chelated complexes formed with all these dihydroxamic acids was found. All the results obtained suggest that one dihydroxamic acid (even the 2,2-DIHA) is able to complete the four coordination sites of a MoO₂ ²⁺ core forming simple mononuclear complexes. Favoured monomeric structures of the bis-chelated complexes of these dihydroxamic acids are also suggested with V(V) having the smallest ionic radius among the three metal ions studied. In the case of iron(III), however, clear indication was obtained for the slightly different complexation behaviour of 2,2-DIHA. Namely, the formation of the mononuclear bis-chelated complex with this shortest ligand seems to have sufficient strain to induce the formation of bimetallic species such as [Fe(2,2-DIHA)₂Fe)]²⁺.     

EPR characterization of alcohol complexes of ferric myoglobin and hemoglobin

Frozen solution electron paramagnetic resonance spectra of the aquo, methanol, and ethanol complexes of ferric myoglobin and hemoglobin are quantitatively analyzed in terms of the rhombic to tetragonal symmetry ratio and the admixture of quartet states, both with regard to central values of these parameters and the widths of their distributions. In both the methanol and ethanol complexes of ferric myoglobin the main change from the aquo complex is a narrowing of the spread in the rhombic to tetragonal symmetry ratio (reduction in structural variation). The alcohol complexes of both the alpha- and beta-chains within the tetramer of ferric hemoglobin are characterized by a lowering of symmetry (as compared with the aquo complex). Qualitative differences in distribution widths among the complexes are consistent with an origin in molecular structure and dynamics rather than in ice matrix-induced strain.     

Ferrylmyoglobin formation induced by acute magnesium deficiency in perfused rat heart causes cardiac failure

The oxidation states in intracellular myoglobin and cytochrome oxidase aa₃ were monitored by reflectatnce spectrophotometry in isoltaed perfused rat hearts subjected to an acutely magnesium deficient environment. After exposure to low extracellular [Mg²⁺]_o (i.e., 0.3 mM) for 30 min, more than 80% of the oxymyoglobin converted to its deoxygenated form. The level of reduced cytochrome oxidase aa₃ also increased about 80% in low [Mg²⁺]_o. the deoxymyoglobin was converted further to a species identified as ferrymyoglobin by its reaction with Na₂S to form ferrous sulfmyoglobin which was optically visible. This process, set into motion by acute Mg deficiency, resulted from a direct accessibility of the exogenous peroxide to the cytosolic protein. The results suggest that a pathway leading to cardiac tissue damage, induced magnesium deficiency, is probably involved in the generation of a ferrylmyoglobin radical which could be prevented by addition of ascorbate, which is known to be a one-electron reductant of this hypervalent form of myglobin. In further studies, we also investigated whether addition of different concentrations of ascorbic acid (AA) to the perfusate could enhance myocardial function after exposure to log [Mg²⁺]_o perfusion. Four concentrations of AA (0.5, 1, 5, 10 mM) were tested, indicate that they exert their effects in a concentration-dependent manner; 1 mM AA was the most effective dose in improving aortic output in a Mg-deficient heart. Ferrylmyoglobin formation was found to be formed considerably before intracellylar release of either creatine phosphokinase or lactic dehydrogenase. These studies may have wide implications as a new mechanisms by which extracellular Mg²⁺ can induce myocardial injury and subsequent cardiac failure.     

31P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

Computer simulation of Zn(II) speciation and effect of Gd(III) on Zn(II) speciation in human blood plasma

The speciation and distribution of Zn(II) and the effect of Gd(III) on Zn(II) speciation in human blood plasma were studied by computer simulation. The results show that, in normal blood plasma, the most predominant species of Zn(II) are [Zn(HSA)] (58.2%), [Zn(IgG)](20.1%), [Zn(Tf)] (10.4%), ternary complexes of [Zn(Cit)(Cys)] (6.6%) and of [Zn(Cys)(His)H] (1.6%), and the binary complex of [Zn(Cys)₂H] (1.2%). When zinc is deficient, the distribution of Zn(II) species is similar to that in normal blood plasma. Then, the distribution changes with increasing zinc(II) total concentration. Overloading Zn(II) is initially mainly bound to human serum albumin (HSA). As the available amount of HSA is exceeded, phosphate metal and carbonate metal species are established. Gd(III) entering human blood plasma predominantly competes for phosphate and carbonate to form precipitate species. However, Zn(II) complexes with phosphate and carbonate are negligible in normal blood plasma, so Gd(III) only have a little effect on zinc(II) species in human blood plasma at a concentration above 1.0×10⁻⁴ M.     

Speciation of N-(phosphonomethyl)glycine (glyphosate) in aqueous solutions containing major components of natural fluids

ABSTRACT

Potentiometric measurements in different media and at different temperatures were performed in order to determine protonation constants of glyphosate and formation constants for Ca²⁺ and Mg²⁺ complexes, together with their dependence on ionic strength and ?H⁰ values. Calorimetric measurements were also performed to determine ?H⁰ of protonation. Potentiometric measurements in mixed Ca²⁺ and Mg²⁺ solution allowed us to determine the formation of a mixed metal species. Thermodynamic parameters reported in this work can be used for studying the complete speciation of glp in electrolyte solutions containing the major ionic components of natural fluids.     

The Effect of Overnight Fasting on the Synthesis of Glycerolipids by Liver Slice

The uptake by liver slices of radioactive acetate, palmitate, stearate, linoleate and glycerol into glycerolipids was compared in fed and fasted (overnight, 16 hr) rats.

The incorporation of l-¹⁴C-acetate into long-chain fatty acids and glycerolipids was depressed by fasting. There was a considerable decrease in the incorporation of 1-¹⁴C-palmitate into triglyceride (TG) and that of l-¹⁴C-stearate into phosphatidylcholine (PC) in fasted liver slices. No such differences were observed with l-¹⁴C-linoleate. The incorporation of l-¹⁴C-glycerol into TG was slightly decreased, whereas that into PC and phosphatidylethanolamine (PE) was increased by fasting.

These observations, together with those with the incorporation of the precursors into molecular species of TG, PC and PE, suggested that the changes in the fatty acid composition of glycerolipids by fasting may be governed by the changes in the availability of acyl moieties as well as in the relative balance of the pathways participating to formation of TG and phospholipids.     

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia

We reported previously that Ascaris suum cytochrome b₅, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b₅ functions as an electron carrier between NADH-mediated cytochrome b₅ reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b₅, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of + 7.2 mV and + 19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b₅ were comparable to that for mammalian hemoglobin and cytochrome b₅; association constants were 0.585 × 10³ M^− 1 and 2.32 × 10³ M^− 1, respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.     

O2 and CO reactions with heme proteins: quantum yields and geminate recombination on picosecond time scales

Picosecond time-resolved absorption spectroscopy and low-temperature studies have been undertaken in order to understand the nature of the intrinsic quantum yields and geminate recombination of carbon monoxide and oxygen to hemoglobin and myoglobin. We find that the photoproduct yields at 40 ps and long times (minutes) after photolysis at 8 K are similar; however, the yield of oxygen photoproducts is 0.4 +/- 0.1 while the yield of carbon monoxide photoproducts is 1.0 +/- 0.1 for both myoglobin and hemoglobin. Measurements in the Soret, near-infrared, and far-IR are used to quantitate the photoproduct yields. These results call into question previous cryogenic kinetic studies of O2 recombination. Significant subnanosecond geminate recombination is observed in oxyhemoglobin down to 150 K, while below 100 K this geminate recombination disappears. The lower photoproduct yields for oxyheme protein complexes can be attributed to both subnanosecond and subpicosecond recombination events which are ligand and protein dynamics dependent.     

Amino acid sequence of myoglobin from the chitonLiolophura japonica and a phylogenetic tree for molluscan globins

Myoglobin was isolated from the radular muscle of the chitonLiolophura japonica, a primitive archigastropodic mollusc.Liolophura contains three monomeric myoglobins (I, II, and III), and the complete amino acid sequence of myoglobin I has been determined. It is composed of 145 amino acid residues, and the molecular mass was calculated to be 16,070 D. The E7 distal histidine, which is replaced by valine or glutamine in several molluscan globins, is conserved inLiolophura myoglobin. The autoxidation rate at physiological conditions indicated thatLiolophura oxymyoglobin is fairly stable when compared with other molluscan myoglobins. The amino acid sequence ofLiolophura myoglobin shows low homology (11–21%) with molluscan dimeric myoglobins and hemoglobins, but shows higher homology (26–29%) with monomeric myoglobins from the gastropodic molluscsAplysia, Dolabella, andBursatella. A phylogenetic tree was constructed from 19 molluscan globin sequences. The tree separated them into two distinct clusters, a cluster for muscle myoglobins and a cluster for erythrocyte or gill hemoglobins. The myoglobin cluster is divided further into two subclusters, corresponding to monomeric and dimeric myoglobins, respectively.Liolophura myoglobin was placed on the branch of monomeric myoglobin lineage, showing that it diverged earlier from other monomeric myoglobins. The hemoglobin cluster is also divided into two subclusters. One cluster contains homodimeric, heterodimeric, tetrameric, and didomain chains of erythrocyte hemoglobins of the blood clamsAnadara, Scapharca, andBarbatia. Of special interest is the other subcluster. It consists of three hemoglobin chains derived from the bacterial symbiont-harboring clamsCalyptogena andLucina, in which hemoglobins are supposed to play an important role in maintaining the symbiosis with sulfide bacteria.