Heme structure in cooperative and noncooperative hemoglobins: study by resonant Raman diffusion]

The modifications of heme sites of hemoglobin, which should occur upon apoprotein alterations (responsible for variations of oxygen affinity), have been examined by Resonnant Raman scattering. The oxygenated (R) and deoxygenated (T) shape of apoprotein do not modify the heme states. The spectral differences between these forms are essentially due to the presence or the absence of the sixth ligand.     

Scapharca dimeric hemoglobin: A new mechanism of information transfer between globin chains

The homodimeric hemoglobin component present in the red cells of the bivalve molluscScapharca inaequivalvis, HbI, is endowed with high cooperativity in ligand binding. This behaviour is in contrast with that of vertebrate hemoglobins in which cooperativity is associated with a tetrameric assembly and the presence of two types of chain. Analysis of the aminoacid sequence and immunological data suggested that the assembly of HbI differed from that characteristic of vertebrate hemoglobins and hence that cooperativity had an unusual structural basis. Indeed the X-ray structures of the carbonmonoxy and deoxy derivatives at 2.4 resolution showed that in HbI the heme carrying E and F helices are not exposed to solvent as in the vertebrate hemoglobin tetramer, but form the subunit interface and bring the two heme groups practically in direct contact through a network of hydrogen bonds. Ligand binding brings about marked structural changes that are limited to the heme environment, whereas quaternary changes are only minor. The structural changes in the heme environment result in alterations in the network of interactions between the heme groups which lead to changes in ligand affinity. In HbI therefore cooperativity in ligand binding is achieved through direct heme-heme communication as opposed to the long range information transfer operative in the vertebrate hemoglobin tetramer.     

The dimeric and co-operative myoglobin ofNassa mutabilis. A peculiar case

The myoglobin present in the radular muscle of the Prosobranchia sea snailNassa mutabilis is a peculiar case among myoglobins. It is a dimer showing co-operative oxygen binding equilibrium curves with pO_{2 1/2}=4.7 mmHg, invariant with pH, and n=1.6. Although the globin is composed of 147 amino acid residues, corresponding to a molecular mass of 15760 D, gel filtration chromatography of the native myoglobin indicate Mr=26000±2000 D. Similarly, acrylamide electrophoretic analyses in SDS and velocity sedimentation indicate a molecular mass of about 13000 D for the denatured globin. The molecule is highly unstable and forms slowly a chromogen when aged or immediately upon oxidation to the ferric state. The visible region of the absorption spectrum of the O₂ or CO liganded myoglobin derivatives indicate an altered heme environment. Circular dichroism analyses confirm this indication showing negative Cotton effects in all regions of the heme absorption spectra of the MbO₂ and MbCO derivatives. Interestingly, the CD spectrum of the oxidised met-form shows a positive band almost symmetrical with respect to that of the MbO₂ derivative. This is similar to what reported for the monomeric hemoglobin ofGlycera dibranchiata for which a reversed heme orientation was proposed. Detailed resonance Raman spectroscopic studies have permitted a more direct investigation of the interactions between the heme and the protein. The proximal Fe-Im bond shows a stretching mode frequency down shifted by 5 cm^–1 with respect to the corresponding band of horse heart myoglobin, in good correlation with the much higher instability ofNassa m. myoglobin and its much lower oxygen affinity. The unusual bond instability finds additional support in a kinetic study in which the myoblogin is mixed with CO in buffered solutions at different pH values. This approach gives evidence that the Fe-Im bond is broken upon lowering the pH, with a pK of 4.0±0.2, the highest among those of deoxy hemoproteins. The rupture of the proximal bond appears to occur with a proton-linked transition showing n=1.8±0.1, again indicating cooperativity between the two subunits. The vinyl and propionate heme substituents show resonance Raman spectroscopic bands indicating different modes of interaction with their environment with respect to other myoglobins. Most interestingly, the vinyl stretching mode frequency, typically a single band, appears split in two bands inNassa m. myoglobin. This splitting is evident in all the investigated derivatives of the myoglobin, indicating that vinyl 2 and 4 are not equivalent in this molecule. A similar splitting has been found so far only inChironomus t.t. hemoglobin.     

Cyclophosphamide-impaired regulation of hepatic heme metabolism

Summary Im male rats hepatic cytochromes b₅ and P-450 were reduced at different times after treatment with cyclophosphamide (CP) (200 mg/kg i.p. for 3 days). In contrast, microsomal heme did not change until 48 h after the last dose of CP, leading to accumulation of heme in a non-cytochromal form. Parallel to the above changes the heme metabolism showed derangement: -aminolaevulinate synthase, the rate-limiting enzyme in heme synthesis, was depressed and heme oxygenase, the enzyme which catalyzes the oxidative degradation of heme, was increased.     

A pivotal role for serotonin (5HT) in the regulation of beta adrenoceptors by antidepressants: reversibility of the action of parachlorophenylalanine by 5-hydroxytroptophan

Summary An acute reduction in the synaptic availability of serotonin (5HT) by p-chlorophenlalanine (PCPA) nullifies the decrease in the density of cortical beta adrenoceptors caused by desipramine (DMI) but does not appreciably alter the attenuation of the norepinephrine (NE) sensitive adenylate cyclase. The analysis of competition-binding curves of [³H]-dihydroalprenolol shows that the affinity of the agonist (–)-isoproterenol for cortical beta adrenoceptors is profoundly reduced following PCPA. This reduction in agonist affinity is enhanced by DMI. Resupplying 5HT by by-passing trptophan hydroxylase inhibition, by administering 5-hydroxytryptophan, converts a DMI non-responsive to a DMI responsive beta adrenoceptor population and shifts the markedly decreased agonist affinity towards the affinity values found in control preparations. The results demonstrate the pivotal role of 5HT in the regulation of the density and agonist affinity characteristics of cortical beta adrenoceptors and contribute to the scientific basis of the serotonin-norepinephrine link hypothesis of affective disorders.Acknowledgments. This work was supported by USPHS grant MH-29228 and the Tennessee Department of Mental Health and Mental Retardation. Present address of L. R. Sterank: NOVA Pharmaceutical Corporation, Baltimore (MD 21228, USA).     

Oxygen consumption of hibernating worker ants Dolichoderus quadripunctatus as a function of temperature and level of population]

The oxygen consumption of "hibernating" ants shows a grouping effect translated by a significant decrease in the level of oxygen consumption as a function of grouping effect. The general relation between oxygen consumption and the living body mass is the same, if the living material belongs to a single animal, or a social community of ants.     

Possible origin of zeaxanthin in the marine sponge,Reniera japonica

As part of a study to clarify the origins of biologically active substances in marine sponges, the carotenoids produced by two species of marine bacteria,Flexibacter sp. strain number DK30213 and DK30223, associated with the marine sponge,Reniera japonica, were investigated. Both bacteria were found to produce zeaxanthin [(3R, 3R)-dihydroxy-,-carotene] which is widely distributed in marine organisms. This carotenoid was also detected in the host sponge, suggesting the transport of zeaxanthin from the microorganisms to the host. As zeaxanthin plays the role of a quencher and scavenger for active species of oxygen, it is presumed that the sponge accumulates the bacterial product as a defense substance against the active oxygen species produced under irradiation by strong sunlight. It is thought that the bacteria are symbionts of the host sponge and act by obtaining the solid substrate and medium needed for settlement and growth from the host, and by producing and transmitting the biologically active substance to the host. Zeaxanthin-producing bacteria are also considered to have potential for practical uses by the aquacultural, pharmaceutical and food industries.     

Higher oxygen affinity of sheep Hb C compared to Hb A and Hb B

Summary Sheep which have Hb A, synthesize the perinatal Hb C in response to severe tissue hypoxia. It is known that Hb A displays a higher oxygen affinity than Hb B. The results of this study indicate that Hb C exhibits an oxygen affinity and a Bohr effect higher than those of Hb A and Hb B.Part of this work was supported by a grant of Ministero Pubblica Istruzione.     

Determination of apparent mean mass of proteins associated with heme in the hemoglobin molecule of Arenicola marina (L.), Annelida, Polychaeta]

Protein and iron concentrations and maximum combined oxygen concentration were measured in the blood of the lugworm Arenicola marina. The calculated mean molecular mass of the heme-associated proteins was higher than that reported for known invertebrate and vertebrate intracellular hemoglobins. The difference is probably due to the presence of polypeptide chains not linked to heme groups in the extracellular annelid hemoglobins.     

Marking out a disciplinary common ground: The role of chemical pedagogy in establishing the doctrine of affinity at the heart of British chemistry

This paper presents a case study that contributes to the current debate among historians of chemistry concerning the role and influence of pedagogy in science. Recently, Bernadette Bensaude-Vincent and her colleagues concluded that in nineteenth-century France, ‘textbooks played an important role in discipline building and in creating theories’.1 ¹A. Garcia-Belmar, B. Bensaude-Vincent and J.R. Bertomeu-Sánchez, ‘The Power of Didactic Writings: French Chemistry Textbooks of the Nineteenth Century’, in Pedagogy and the Practice of Science: Historical and Contemporary Perspectives, edited by D. Kaiser (Cambridge, MA, 2005), 243. Developing this idea further, this paper examines the dissemination of knowledge through face-to-face chemical lectures, showing that the influence of pedagogical strategy on theoretical content of the science is far from negligible. The pedagogy of William Cullen was essentially responsible for the prevalence of the doctrine of affinity in British chemistry from the 1760s onwards. Cullen used his affinity theory as a pedagogical tool that to a large extent defined his discipline, and the pedagogical pyramid that he headed similarly ensured that the doctrine would remain at the heart of British chemistry. From a pedagogical tool, the doctrine of affinity was transformed over time into a chemical tool, offering British chemists a disciplinary common ground that both set and reinforced the boundaries to their discipline.     

Crystal structure of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> 908R class C β-lactamase bound to iodo-acetamido-phenyl boronic acid,a transition-state analogue

The structures of the class C -lactamase from Enterobacter cloacae 908R alone and in complex with a boronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 Å, respectively. The structure of the enzyme resembles those of other class C -lactamases. The structure of the complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C -lactamase in complex with a transition-state analogue provides further insights into the mechanism of action of these hydrolases.Received 16 May 2003; accepted 4 June 2003     

La biologie des hématinoprotéides oxygénables

Summary The author reviews a series of biological aspects of the study of oxygenable hematinoproteids, particularly with respect to evolution and adaptation. After a statement of some fundamental concepts of comparative biochemistry and of possible evolutionary relations between oxidation catalysts and oxygen carriers, the natural distribution of hemoglobins is reviewed and their specific characters are enumerated.A review is made of the data relating to the shape of the oxygen-dissociation curves and to the affinity of hæmoglobin for oxygen. It appears, generally speaking, that hyperbolic or almost hyperbolic curves and high affinity for oxygen are characteristic of primitive or embryonic hæmoglobins.A study of the function of hæmoglobin in the respiratory cycle of several animal species shows the vital importance of the oxygen carrier as well as the adaptation of the shape and position of the dissociation curve to the character of the respiratory function in the animal considered.The function of hæmoglobin in invertebrates, as oxygen carrier as well as providing a store of oxygen, is emphasized by a review of experimental data.The function of oxygen in the transport of carbon dioxide is reviewed from the standpoint of comparative biochemistry, and the lack of our knowledge is deplored.The characteristics of chlorocruorin show it to be a chemical mutation of an Annelid hæmoglobin.Our lack of knowledge in the field of the comparative biochemistry of hæmoglobin and chlorocruorin metabolism is pointed out.

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Conférence principale, présentée à la Société suisse de biologie médicale lors de la 127^e Assemblée générale de la Société helvétique des sciences naturelles à Genève, le 31 août 1947.     

The bacterial SoxAX cytochromes

SoxAX cytochromes are heme-thiolate proteins that play a key role in bacterial thiosulfate oxidation, where they initiate the reaction cycle of a multi-enzyme complex by catalyzing the attachment of sulfur substrates such as thiosulfate to a conserved cysteine present in a carrier protein. SoxAX proteins have a wide phylogenetic distribution and form a family with at least three distinct types of SoxAX protein. The types of SoxAX cytochromes differ in terms of the number of heme groups present in the proteins (there are diheme and triheme versions) as well as in their subunit structure. While two of the SoxAX protein types are heterodimers, the third group contains an additional subunit, SoxK, that stabilizes the complex of the SoxA and SoxX proteins. Crystal structures are available for representatives of the two heterodimeric SoxAX protein types and both of these have shown that the cysteine ligand to the SoxA active site heme carries a modification to a cysteine persulfide that implicates this ligand in catalysis. EPR studies of SoxAX proteins have also revealed a high complexity of heme dependent signals associated with this active site heme; however, the exact mechanism of catalysis is still unclear at present, as is the exact number and types of redox centres involved in the reaction.     

The 2.4-A crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin

Penicillin-binding proteins (PBPs) are membrane proteins involved in the final stages of peptidoglycan synthesis and represent the targets of beta-lactam antibiotics. Enterococci are naturally resistant to these antibiotics because they produce a PBP, named PBP5fm in Enterococcus faecium, with low-level affinity for beta-lactams. We report here the crystal structure of the acyl-enzyme complex of PBP5fm with benzylpenicillin at a resolution of 2.4 A. A characteristic of the active site, which distinguishes PBP5fm from other PBPs of known structure, is the topology of the loop 451-465 defining the left edge of the cavity. The residue Arg464, involved in a salt bridge with the residue Asp481, confers a greater rigidity to the PBP5fm active site. In addition, the presence of the Val465 residue, which points into the active site, reducing its accessibility, could account for the low affinity of PBP5fm for beta-lactam. This loop is common to PBPs of low affinity, such as PBP2a from Staphylococcus aureus and PBP3 from Bacillus subtilis. Moreover, the insertion of a serine after residue 466 in the most resistant strains underlines even more the determining role of this loop in the recognition of the substrates.     

Methemoglobin formation and reduction in relation to hemoglobin oxygen affinity

Summary It is suggested that although the high oxygen affinity hemoglobin (Hb LR) (143(H21)HisGln) with stabilization of R quaternary conformation is not more susceptible to the oxidizing effect of nitrites in vitro, in an in vivo situation where hemoglobin is partly deoxygenated, it might be more susceptible to methemoglobin formation due to higher oxidation and lower reduction rate.Supported by NIH grant No. 5 RO1 AM 20181-02 and VA grant No. 5455-002.Acknowledgments. The technical assistance of Ms Carol A. Perry is highly appreciated.     

Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory waspphilanthus triangulum F

Summary Two components of the venom of the predatory waspPhilanthus triangulum F. significantly reduce — to a greater or less extent — the high affinity uptake of glutamate in rat hippocampus. A concentration of 10 M -PTX caused a reduction of 74%, while the other component, -PTX, at the same concentration, caused a reduction of 18%. Hence the effect of -PTX on high affinity glutamate uptake in the hippocampus is comparable with its effect on high affinity glutamate uptake in insect neuromuscular junctions. Contrary to our previous findings that -PTX has no effect on high affinity glutamate uptake in insect glutamatergic terminal axons, however, -PTX significantly reduces high affinity glutamate uptake in the hippocampus, albeit less effectively than -PTX.     

血红素蛋白结构及功能的相互转换

本文综述了利用蛋白质工程方法,实现不同血红素蛋白之间结构及功能的相互转换,包括氧载体与电子传递蛋白间的相互转换、氧载体与生物催化剂间的相互转换、电子传递蛋白与生物催化剂间的相互转换,以及不同类型血红素蛋白之间的相互转换。这些成功的研究成果,丰富了我们对金属蛋白结构-性质-反应-功能之间精妙关系的深入认识。     

Nature of the FeO2 bonding in myoglobin: An overview from physical to clinical biochemistry

Summary The iron(II)-dioxygen bond in myoglobin and hemoglobin is a subject of wide interest. Studieas range from examinations of physical-chemical properties dependent on electronic structure, to investigations of stability as a function of oxygen supply. Stability properties are of particular importance in vivo, since the oxygenated form is known to be oxidized easily to the ferric form, which cannot be oxygenated and is therefore physiologically inactive.Kinetic and thermodynamic studies of the stability of native oxymyoglobin have revealed a new feature in FeO₂ bonding. In vivo. the iron center is always subject to a nucleophilic attack of the water molecule or hydroxyl ion, which can enter the heme pocket from the surrounding solvent, and thereby irreversibly displace the bound dioxygen from MbO₂ in the form of O ₂ ^– so that the iron is converted to the ferric form. A free energy diagram for the potential reactions of FeO₂ visualizes myoglobin as a molecular structure that can provide in solution the delicate balance of kinetic and thermodynamic factors necessary to stabilize reversible oxygenation, as opposed to irreversible autoxidation to metmyoglobin.     

High and low affinity transport ofL-arginine in rat brain synaptosomes

The uptake ofL-arginine into purified rat brain synaptosomes was investigated with respect to time and various concentrations ofL-[³H] arginine. Specific uptake was found to be linear with time for up to 5 min of incubation at 37°C. Electrolytes, including sodium chloride, potassium chloride, magnesium chloride and calcium chloride, inhibited uptake of 3 ML-arginine, and the inhibitory effect increased with increased electrolyte concentration under constant osmolarity. It was found thatL-arginine was transported into synaptosomes by two uptake components — a high affinity component (3.5 M) and a low affinity component (100 M). These two components were similar to the Ly⁺ system because of their extreme sensitivity to inhibition byL-lysine andL-ornithine but were distinguishable from each other by kinetic analysis of the uptake data and by their relative sensitivity to inhibition by several amino acids.