The flash-photolysis study of recombination of hemoproteins with carbon monoxide at low temperatures]

The hemoproteins (sperm whale myoglobin, hemoglobin from larvae of Chironomus thummi thummi, bovine hemoglobin) were studied in viscous solvents (saturated sucrose solution, glycerol and water-glycerol solutions) in the temperature range +50 divided by -100 degrees C. At low temperatures the three-phase kinetics of Mb recombination with CO was observed. The velocities of two "fast" reactions did not depend on ligand concentration. This fact indicates that they are due to a so called cage-effect. The formation of the cage is caused apparently by a local change of the solvent state in the heme region. To explain the biphasic "cage" kinetics it has been assumed that during some time after photodissociation myoglobin remains in the "ligand-bound" conformation and reacts with CO faster than the "normal" myoglobin. For other hemoproteins the "cage-effect" was not observed. For all the studied hemoproteins the quantum yield of photodissociation decreased as the temperature decreased. The decrease of quantum yield can be described by the Arrenius law. The rates of the decrease of quantum yield differ for different proteins.     

Effects of 2,4-substituents of deuteroheme upon peroxidase functions. Reactions of peroxidase and myoglobin with oxygen, carbon monoxide and alkylisocyanides

The oxygen affinity of myoglobins increased in the order diacetyl- < chlorocruoro- < proto- < meso-myoglobins, which was a decreasing order of electron-withdrawing capacities of 2,4-substituents of deuteroheme. The results led to a conclusion that the π bonding predominates over the σ bonding in the myoglobin-oxygen interaction.On the contrary, the affinities of myoglobins and reduced peroxidases for ethylisocyanide decreased as the electron-withdrawing capacities of the substituents were decreased and it was therefore concluded that the σ bonding plays a dominant role in the ethylisocyanide binding to these hemoproteins.No simple relationship was observed in the cases of reduced peroxidase-CO complexes and myoglobin-isopropylisocyanide and -tertiarybutylisocyanide complexes. Among the hemoproteins tested, protohemoproteins appeared to have the lowest affinities for these ligands.As was the case with ligands such as azide and cyanide, the affinities of a deuterohemo-protein for oxygen, CO, and isocyanides were invariably much higher than the value expected from the Hammett-type relationship. The linear relationship between enthalpy and entropy was found to be applied for the reactions of ligands and hemoproteins including deutero derivatives and the irregularly high affinities of deuterohemoproteins for ligands could not be explained on the basis of thermodynamic analyses.     

Mechanism of detergent interaction with hemoproteins in aqueous media

The interactions of myoglobin, cytochrome c, and cytochrome P-450 LM-2 isolated from rabbit liver microsomes with detergents of type A (TM 3-12, Tween 20, Triton N-101) and detergent of B type (sodium cholate) in aqueous media were studied. These interactions are accompanied by a decrease in the Soret band intensity for all three hemoproteins. The rate of this process depends on the nature and concentration of the detergent as well as on temperature. The rate of the Soret band decrease is maximal for the zwitter-ionic surfactant TM 3-12. The rate constants of hemoprotein transformation depend on the detergent concentration. The detergent effects on the conformation and structure of the protein were demonstrated, using CD spectra and second derivatives of the absorption spectra of the hemoproteins in the presence of the detergents. The activation energies for myoglobin transformation in the presence of various detergents are equal to 17-23 kcal/mol and possibly reflect the cleavage of the coordinative heme-apoprotein bonds. A model of detergent interaction with hemoproteins is discussed. According to this model, the bimolecular interaction of the proteins with surfactants is observed at the detergent concentrations that are much lower than those for critical micelle formation values.     

Applications of electron paramagnetic resonance to the study of biological molecules]

In a first part, an elementary theoretical introduction to ESR is given. In a second part, selected examples illustrate the possibilities of this method. Studies of hemoproteins, flavoproteins, model complexes and phospholipid systems are described. This review is made for biochemists who have no experience in ESR.     

A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol.

Measurement of myeloperoxidase (MPO; EC 1.11.1.7) activity is often used as a marker of neutrophil infiltration into tissues. However, most enzymatic assays for MPO are susceptible to interference from other peroxidases (including eosinophil peroxidase, EPX) and hemoproteins (such as hemoglobin and myoglobin) present in the tissues. In this report, we describe a bromide-dependent chemiluminescence (Br-CL) assay that uses luminol as a chemiluminescence probe. The assay can distinguish between MPO and nonspecific peroxidase reactions. The MPO-specific reaction is believed to proceed in two steps: (i) the enzymatic generation of hypobromous acid (HOBr) from KBr and H(2)O(2) at pH 5 and (ii) the spontaneous reaction of HOBr and H(2)O(2) with luminol to give a Br-CL signal. The assay is sufficiently sensitive to allow detection of MPO in <100 human neutrophils. Other peroxidases and hemoproteins do not interfere with the Br-CL signal. Although EPX can also oxidize bromide to generate HOBr, activities of MPO and EPX can be distinguished at different pHs. As a demonstration of the utility of the Br-CL assay, MPO activity was measured in murine tumors known to be infiltrated with neutrophils. A statistically significant correlation was seen between MPO activity and histological neutrophil counts in the tumors (r = 0.69, P < 0.01, n = 14). The assay should have wide application for measuring the neutrophil content of tissues.     

Drug metabolism in the newborn

The duration and intensity of drug action depend not only on the dose of the drug but also on the rates at which drugs are transformed to products that can be excreted readily by the kidney. Two general categories of drug metabolism occur in the liver: phase 1 reactions (oxidations-reductions and hydrolyses) and phase 2 reactions (synthetic conjugations). Phase 1 reactions produce functional groups that can participate in phase 2 reactions. Phase 1 reactions are almost nonexistent in the fetuses of laboratory animals; however, many appear in primates during the first trimester of gestation. Phase 2 reactions are deficient prenatally in both rodents and primates. Parturition triggers a surge of both phase 1 and phase 2 reactions. The lack of uniformity of the development of phase 1 oxidative reactions during the early neonatal period reflects the multiplicity of cytochrome P-450 hemoproteins, the terminal oxidases responsible for most hepatic oxidative biotransformations. The rate of recovery of chemically induced losses of cytochrome P-450 systems is age dependent.     

Activation mechanism of prostaglandin endoperoxide synthetase by hemoproteins

The mechanism of the activation of prostaglandin endoperoxide synthetase by hemeproteins was investigated using the enzyme purified from bovine seminal vesicle microsomes. At pH 8, the maximal enzyme activities with methemoglobin (2 microM), indoleamine 2,3-dioxygenase (2 microM), and metmyoglobin (2 microM) were 70%, 42%, and 15% of that with 1 microM hematin. Apomyoglobin and apohemoglobin inhibited the enzyme activities caused by hemoproteins as well as that caused by hematin. The inhibition was removed by the addition of excess hematin. The dissociation of heme from hemoproteins was demonstrated by trapping the free heme with human albumin or to a DE-52 column. The dissociation of heme from methemoglobin was facilitated by increasing concentrations of arachidonic acid. The amount of heme dissociated from hemoproteins (methemoglobin, metmyoglobin, and indoleamine 2,3-dioxygenase) in the presence of arachidonic acid correlated with their stimulatory effects on the prostaglandin endoperoxide synthetase activity. Horseradish peroxidase and beef liver catalase, the hemes of which were not dissociated in the presence of arachidonic acid, were ineffective in activating prostaglandin endoperoxide synthetase. Spectrophotometric titration of prostaglandin endoperoxide synthetase with hematin demonstrated that the enzyme bound hematin at the ratio of 1 mol/mol with an association constant of 0.6 x 10(8) M-1. From these results, we conclude that hemoproteins themselves are ineffective in activating prostaglandin endoperoxide synthetase and free hematin dissociated from the hemoproteins by the interaction of arachidonic acid is the activating factor for the enzyme.     

Hemin Replaces Serum as a Growth Requirement for Naegleria

Four strains of Naegleria gruberi were grown axenically without serum. Serum was replaced by hemin or two selected hemoproteins. Aside from the utility of eliminating serum from the culture medium, the present work shows that Naegleria does not require intact protein, and establishes a specific micronutrient requirement for this amoebo-flagellate.     

Cytochrome P450 systems--biological variations of electron transport chains

Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.     

Cytochrome P450 systems—biological variations of electron transport chains

Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.     

The green hemoproteins of bovine erythrocytes. II. Spectral, ligand-binding, and electrochemical properties

The two green hemoproteins isolated from bovine erythrocytes (form I and form II) have been characterized as to spectral, electrochemical, and chemical properties. The absorption spectra of the isolated hemoproteins are typical of high spin ferric states. Reduction of the hemoproteins yields high spin ferrohemoproteins. Complexation of the ferrohemoproteins with CO and the ferrihemoproteins with cyanide yields low spin complexes, demonstrating the presence of an exchangeable weak field ligand in both the ferrous and ferric states of the hemoproteins. The differences in position and intensity of the absorption peaks of the visible spectra allow the two forms to be distinguished from one another. The midpoint potential of forms I and II were found to be +0.075 and +0.019 V, respectively, at pH 6.4 and +0.038 and -0.005 V, respectively, at pH 7.0. This is consistent with the gaining of 1 proton/electron during the reduction. The Nernst plot reveals an unusual 0.5-electron transfer, whereas a quantitative titration demonstrates a 1-electron transfer. Form I binds cyanide more tightly than form II (KD of 84 and 252 micrometer, respectively). The observed spectral, electrochemical, and ligand-binding differences between forms I and II can be explained in terms of a greater electron-withdrawing ability of the side chains of the heme of form I relative to the heme of form II.     

Guanylate cyclase from bovine lung. Evidence that enzyme activation by phenylhydrazine is mediated by iron-phenyl hemoprotein complexes

The mechanism of activation of soluble guanylate cyclase purified from bovine lung by phenylhydrazine is reported. Heme-deficient and heme-containing forms of guanylate cyclase were studied. Heme-deficient enzyme was activated 10-fold by NO but was not activated by phenylhydrazine. Catalase or methemoglobin enabled phenylhydrazine to activate guanylate cyclase 10-fold and enhanced activation by NO to over 100-fold. Heme-containing enzyme was activated only 3-fold by phenylhydrazine but over 100-fold by NO. Added hemoproteins enhanced enzyme activation by phenylhydrazine to 12-fold without enhancing activation by NO. Reducing or anaerobic conditions inhibited, whereas oxidants enhanced enzyme activation by phenylhydrazine plus catalase, and KCN had no effect. In contrast, enzyme activation by NO and NaN3 was inhibited by oxidants or KCN. NaN3 required native catalase, whereas phenylhydrazine also utilized heat-denatured catalase for enzyme activation. Thus, the mechanism of guanylate cyclase activation by phenylhydrazine differed from that by NO or NaN3. Guanylate cyclase activation by phenylhydrazine resulted from an O2-dependent reaction between phenylhydrazine and hemoproteins to generate stable iron-phenyl hemoprotein complexes. These complexes activated guanylate cyclase in the absence of O2, but lost activity after acidification, basification, or heating. Gel filtration of prereacted mixtures of [U-14C]phenylhydrazine plus hemoproteins resulted in co-chromatography of radioactivity, protein, and guanylate cyclase stimulating activity, and yielded a phenyl-hemoprotein binding stoichiometry of four under specified conditions (one phenyl/heme). [14C]Phenyl bound to heme-containing but not heme-deficient guanylate cyclase and binding correlated with enzyme activation. Moreover, reactions between enzyme and iron-[14C] phenyl hemoprotein complexes resulted in the exchange or transfer of iron-phenyl heme to guanylate cyclase and this correlated with enzyme activation.     

Picosecond kinetics of cytochromes b5 and c

Ligand photolysis and subsequent recombination in cytochromes b5 and c have been studied with picosecond resolution. In both proteins, an iron-histidine bond is broken after excitation with 314-nm light, and recombination occurs with a rate constant of about 1.4 x 10(11) s-1. Photolysis and reformation of the iron-histidine bond may be surprising as these hemoproteins do not reversibly bind ligands in nature. The findings are explained using results both from experiments on model hemes and from computer investigations with atomic resolution on the three-dimensional structure of the protein. After photolysis, the formation and recombination of the geminate contact pair are attributed to simple low amplitude ligand bond rotations, a result that can be applied to geminate processes in other hemoproteins and model heme compounds as well.     

Quantitative Radiation Effects in Hemoproteins and their Constituents

In order to verify the “radiation protection” effect of prosthetic groups in irradiated conjugated proteins and the influence of their electronic structure on intramolecular excitation transfer, two series of substances were examined. The first was composed of ferrihemoglobin and its constituents and the second was composed of similar hemoproteins with heme groups with major differences in electronic structure. The results point to a strong radiation protection effect of the heme groups and a dependence of radical yield of hemoproteins on the electronic structure of the heme. A possible interpretation of this latter effect is discussed.     

Oxidation of low-density lipoprotein by hemoglobin-hemichrome

Hemoglobin and myoglobin are inducers of low-density lipoprotein oxidation in the presence of H(2)O(2). The reaction of these hemoproteins with H(2)O(2) result in a mixture of protein products known as hemichromes. The oxygen-binding hemoproteins function as peroxidases but as compared to classic heme-peroxidases have a much lower activity on small sized and a higher one on large sized substrates. A heme-globin covalent adduct, a component identified in myoglobin-hemichrome, was reported to be the cause of myoglobin peroxidase activity on low-density lipoprotein. In this study, we analyzed the function of hemoglobin-hemichrome in low-density lipoprotein oxidation. Oxidation of lipids was analyzed by formation of conjugated diene and malondialdehyde; and oxidation of Apo-B protein was analyzed by development of bityrosine fluorescence and covalently cross-linked protein. Hemoglobin-hemichrome has indeed triggered oxidation of both lipids and protein, but unlike myoglobin, hemichrome has required the presence of H(2)O(2). In correlation to this, we found that unlike myoglobin, hemichrome formed by hemoglobin/H(2)O(2) does not contain a globin-heme covalent adduct. Nevertheless, hemoglobin-hemichrome remains oxidatively active towards LDL, indicating that other components of the oxidatively denatured hemoglobin should be considered responsible for its hazardous activity in vascular pathology.     

Molecular characteristics of many hemoproteins: a survey of molecular weights, sedimentation coefficients, other molecular parameters and amino acid compositions

Data on molecular weights, sedimentation coefficients, other molecular parameters and amino acids compositions of many hemoproteins were collected from the literature and studied. The results of the survey gave a general view of the molecular characteristics of hemoproteins and also revealed the presence of various statistical correlations among the molecular parameters and amino acid compositions. Some of the correlations were found to be practically useful for the estimation of number of heme per molecule, molecular weight or partial specific volume. Discussions were made on the possible structural basis of the molecular characteristics of hemoproteins.     

Structural and thermodynamic consequences of b heme binding for monomeric apoglobins and other apoproteins

The binding of a cofactor to a protein matrix often involves a reorganization of the polypeptide structure. b Hemoproteins provide multiple examples of this behavior. In this minireview, selected monomeric and single b heme proteins endowed with distinct topological properties are inspected for the extent of induced refolding upon heme binding. To complement the data reported in the literature, original results are presented on a two-on-two globin of cyanobacterial origin (Synechococcus sp. PCC 7002 GlbN) and on the heme-containing module of FixL, an oxygen-sensing protein with the mixed alpha/beta topology of PAS domains. GlbN had a stable apoprotein that was further stabilized and locally refolded by heme binding; in contrast, apoFixLH presented features of a molten globule. Sequence analyses (helicity, disorder, and polarity) and solvent accessibility calculations were performed to identify trends in the architecture of b hemoproteins. In several cases, the primary structure appeared biased toward a partially disordered binding pocket in the absence of the cofactor.     

Hemoglobin and myoglobin embedded in dry poly(vinyl alcohol) film for X-ray absorption studies

Solid hemoprotein samples are prepared by embedding proteins in thin poly(vinyl alcohol) films. These film samples have several unique qualities: in the solid poly(vinyl alcohol) matrix, hemoproteins have the same properties as in frozen buffer solution (proven by optical absorption, ligand recombination kinetics and EXAFS); they are very stable, easy to store and resistant to radiation; damage; protein concentration can be as high as 15 mM; light transparency is as good as liquid solution samples; and they can be made as thin as 20 microns, so that 100% photolysis across a film, even with a high protein concentration, is easily achievable. The film samples are ideal for X-ray studies of optically illuminated hemoproteins.     

The oscillator strengths of hemoproteins. Their relation to the coordination structure and magnetic susceptibility

The oscillator strengths of hemoproteins in the light frequency range of 1.11 X 10(4) to 3.23 X 10(4) cm-1 (wavelength range of 900 to 310 nm) were measured by means of computer-assisted spectrophotometry. The obtained values of oscillator strength per molar heme ranged from about 1.4 to 2.2. By comparing the oscillator strength values of the ferric and ferric cyanide-bound forms of hemoproteins and also the values of low molecular weight ferric heme complexes, it was found that the oscillator strength was lower for those hemoproteins whose heme was coordinated with strong field ligands. It was also found that the hemoproteins showing a smaller pH-dependent change in the carbon monoxide-difference spectrum had lower oscillator strengths. The following linear relation was observed, with various ligand complexes of bovine methemoglobin, horse metmyoglobin, and ferric horseradish peroxidase, between the oscillator strength (f) determined in the present study and the respective magnetic susceptibility (10(6) X chi 20 degrees M) values in the literature: f = A (10(6) X chi 20 degrees M) + B. The values of constants A and B in the equation were estimated for horseradish peroxidase, methemoglobin, and metmyoglobin. On varying the temperature in the range of 0 to 40 degrees C, the oscillator strength of the metmyoglobin-azide complex changed in parallel with the change in the spin state. Taking advantage of the fact that fluoride complexes of many hemoproteins show 10(6) X chi 20 degrees M values close to 14,500 and also that the values of intersection B are around 86.4% of the respective values of the fluoride complexes of ferric horseradish peroxidase, methemoglobin, and metmyoglobin, an empirical equation was evolved for the calculation of an approximate 10(6) X chi 20 degrees M value from the f value of a given complex (fobs) and that of the fluoride complex (fF) of a hemoprotein. The approximate magnetic susceptibilities of various ligand complexes of bovine lactoperoxidase could be thus calculated with the equation. The oscillator strengths of ferrous hemoproteins were also investigated and ligand-dependent regular changes were found.