Jonathan B. Wittenberg

Myoglobin function reassessed

SUMMARY The heart and those striated muscles that contract for long periods, having available almost limitless oxygen, operate in sustained steady states of low sarcoplasmic oxygen pressure that resist change in response to changing muscle work or oxygen supply. Most of the oxygen pressure drop from the erythrocyte to the mitochondrion occurs across the capillary wall. Within the sarcoplasm, myoglobin, a mobile carrier of oxygen, is developed in response to mitochondrial demand and augments the flow of oxygen to the mitochondria. Myoglobin-facilitated oxygen diffusion, perhaps by virtue of reduction of dimensionality of diffusion from three dimensions towards two dimensions in the narrow spaces available between mitochondria, is rapid relative to other parameters of cell respiration. Consequently, intracellular gradients of oxygen pressure are shallow, and sarcoplasmic oxygen pressure is nearly the same everywhere. Sarcoplasmic oxygen pressure, buffered near 0.33 kPa (2.5 torr; equivalent to approximately 4 μmol l-1 oxygen) by equilibrium with myoglobin, falls close to the operational Km of cytochrome oxidase for oxygen, and any small increment in sarcoplasmic oxygen pressure will be countered by increased oxygen utilization. The concentration of nitric oxide within the myocyte results from a balance of endogenous synthesis and removal by oxymyoglobin-catalyzed dioxygenation to the innocuous nitrate. Oxymyoglobin, by controlling sarcoplasmic nitric oxide concentration, helps assure the steady state in which inflow of oxygen into the myocyte equals the rate of oxygen consumption.

Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide

Mycobacterium tuberculosis, the causative agent of human tuberculosis, and Mycobacteriumbovis each express two genes, glbN and glbO, encoding distantly related truncated hemoglobins (trHbs), trHbN and trHbO, respectively. Here we report that disruption of M. bovis bacillus Calmette–Guérin glbN caused a dramatic reduction in the NO-consuming activity of stationary phase cells, and that activity could be restored fully by complementing knockout cells with glbN. Aerobic respiration of knockout cells was inhibited markedly by NO in comparison to that of wild-type cells, indicating a protective function for trHbN. TyrB10, which is highly conserved in trHbs and interacts with the bound oxygen, was found essential for NO consumption. Titration of oxygenated trHbN (trHbN⋅O2) with NO resulted in stoichiometric oxidation of the protein with nitrate as the major product of the reaction. The second-order rate constant for the reaction between trHbN⋅O2 and NO at 23°C was 745 μM−1⋅s−1, demonstrating that trHbN detoxifies NO 20-fold more rapidly than myoglobin. These results establish a role for a trHb and demonstrate an NO-metabolizing activity in M. tuberculosis or M. bovis. trHbN thus might play an important role in persistence of mycobacterial infection by virtue of trHbN′s ability to detoxify NO.

Expression, Purification, and Properties of Recombinant Barley (Hordeum sp.) Hemoglobin OPTICAL SPECTRA AND REACTIONS WITH GASEOUS LIGANDS

A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s−1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.

Chlamydomonas Chloroplast Ferrous Hemoglobin HEME POCKET STRUCTURE AND REACTIONS WITH LIGANDS

We report the optical and resonance Raman spectral characterization of ferrous recombinantChlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>107 μm −1 s−1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s−1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm−1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.

The purification, characterization and ligand-binding kinetics of hemoglobins from root nodules of the non-leguminous Casuarina glauca — Frankia symbiosis

The presence of a membrane-bound hemoglobin in aqueous extracts of nitrogen-fixing Casuarina root nodules (Davenport, H.E. (1960) Nature 186, 653–654) has been confirmed. By strictly anaerobic grinding and extraction under carbon monoxide, with inclusion of soluble polyvinylpyrrolidone and zwitterionic detergent in extraction buffer, soluble carboxyhemoglobin was obtained. This was purified by anaerobic ‘adsorption’ chromatography on Sephacryl S-200 (Pharmacia) followed by aerobic molecular exclusion chromatography on Sephadex G-75 (Pharmacia) to yield very stable oxyhemoglobin. By preparative-scale isoelectric focusing Casuarina oxyhemoglobin is separable into three major components comprising approx. 20% of applied protein, and very many minor components. Monomeric Casuarina hemoglobin is similar to other plant hemoglobins in respect of molecular weight (≈ 17 500), optical spectra, extremely rapid kinetics of binding to oxygen and carbon monoxide and high oxygen affinity (P50 ≈ 0.074 torr). Hence, it is possible that this protein functions in the Casuarina symbiosis as does leghemoglobin in leguminous nitrogen-fixing symbioses. Western blot analysis showed close immunological relationships between the non-leguminous Casuarina and Parasponia hemoglobins and a weaker relationship between these two proteins and soybean leghemoglobin. It is proposed that these hemoglobins from widely separated plant orders have a common evolutionary origin.

Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: separation from hemoglobin by subunit-exchange chromatography.

Abstract Myoglobin is extracted from muscle and separated from blood hemoglobin by subunit-exchange chromatography on a column of Sepharose 4B to which hemoglobin α-β subunits are linked covalently. Hemoglobin is retained on the column. Myoglobin in the effluent is determined spectrophotometrically as ferrous myoglobin or as carbon monoxide ferrous myoglobin. The method is applicable to cardiac, smooth, or skeletal muscle from mammals, reptiles, birds, and teleost fish, but failed with the one amphibian and the one shark tested.

Cyanide Binding to Lucina pectinata Hemoglobin I and to Sperm Whale Myoglobin: An X-Ray Crystallographic Study

The x-ray crystal structures of the cyanide derivative of Lucina pectinata monomeric hemoglobin I (L. pectinata HbI) and sperm whale (Physeter catodon) myoglobin (Mb), generally taken as reference models for monomeric hemoproteins carrying hydrogen sulfide and oxygen, respectively, have been determined at 1.9 A (R-factor = 0. 184), and 1.8 A (R-factor = 0.181) resolution, respectively, at room temperature (lambda = 1.542 A). Moreover, the x-ray crystal structure of the L. pectinata HbI:cyanide derivative has been studied at 1.4-A resolution (R-factor = 0.118) and 100 K (on a synchrotron source lambda = 0.998 A). At room temperature, the cyanide ligand is roughly parallel to the heme plane of L. pectinata HbI, being located approximately 2.5 A from the iron atom. On the other hand, the crystal structure of the L. pectinata HbI:cyanide derivative at 100 K shows that the diatomic ligand is coordinated to the iron atom in an orientation almost perpendicular to the heme (the Fe-C distance being 1.95 A), adopting a coordination geometry strictly reminescent of that observed in sperm whale Mb, at room temperature. The unusual cyanide distal site orientation observed in L. pectinata HbI, at room temperature, may reflect reduction of the heme Fe(III) atom induced by free radical species during x-ray data collection using Cu Kalpha radiation.

Siderophore-bound iron in the peribacteriod space of soybean root nodules

Water-soluble, non-leghemoglobin iron (125 µmol kg-1 wet weight nodule) is found in extracts of soybean root nodules. This iron is probably confined to the peribacteroid space of the symbiosome, where its estimated concentration is 0.5 – 2.5 mM. This iron is bound by siderophores (compounds binding ferric iron strongly) which are different for each of the three strains of Bradyrhizobium japonicum with which the plants were inoculated. One of these, that from nodules inoculated with strain CC 705, is tentatively identified as a member of the pseudobactin family of siderophores.Leghemoglobin is present in only very small amounts in the peribacteroid space of symbiosomes isolated from soybean root nodules, and may be absent from the peribacteroid space of the intact nodule.

[2] Preparation of myoglobins

Publisher Summary This chapter presents procedures for the isolation of intracellular oxygen-binding proteins of tissues, called “tissue hemoglobins” in the widest sense. All of these, except Ascaris and yeast hemoglobin, are monomers or dimers having a minimum molecular weight of 18,000 with similar optical spectra and chemical reactivity. Strictly, only muscle hemoglobin should be called “myoglobin”; by extension the term is often applied to other tissue hemoglobins as well. Ferric myoglobin may be purified by chromatography on carboxymethyl (CM) cellulose, usually at slightly acid pH or on diethylaminoethyl (DEAE) cellulose. The choice of preparative procedure depends on the use to which the purified myoglobin will be put. Both DEAE and CM ion-exchange columns yield myoglobin that is pure in the sense of being free from contaminating polypeptide chains. Better resolution of forms of myoglobin differing only in charge is achieved on CM-cellulose. Such columns, however, are usually operated at acid pH, and it is a matter of experience that oxymyoglobin exposed to mildly acidic conditions becomes ferric and, in the process, undergoes some minor but apparently irreversible change. The chapter also explains the isolation and purification of vertebrate myoglobins.

The hemoglobin of Ascaris perienteric fluid. I. Purification and spectra.

Abstract 1. 1. The hemoglobin of Ascaris lumbricoides perienteric fluid has been The product is monodisperse in the analytical ultracentrifuge and when examined by electrophoresis on paper or in a bed of starch granules. 2. 2. The sedimentation coefficient, s 20 , w of the hemoglobin is 11.8 S. 3. 3. The minimum molecular weight per mole heme was estimate4d to be 40600 from both dry weight and amino acid composition. 4. 4. The absorption spectra of hemoglobin derivatives were re-examined and the extinction coefficients determined.