Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish.

Myoglobin (Mb) buffers intracellular O2 and facilitates diffusion of O2 through the cell. These functions of Mb will be most effective when intracellular PO2 is near the partial pressure of oxygen at which Mb is half saturated (P50) of the molecule. We test the hypothesis that Mb oxygen affinity has evolved such that it is conserved when adjusted for body temperature among closely related animals. We measure oxygen P50s tonometrically and oxygen dissociation rate constants with stopped flow and generate amino acid sequence from cDNA of Mbs from fish with different body temperatures. P50s for the endothermic bluefin tuna, skipjack tuna, and blue marlin at 20 degrees C were 0.62 +/- 0.02, 0.59 +/- 0.01, 0.58 +/- 0.04 mmHg, respectively, and were significantly lower than those for ectothermic bonito (1.03 +/- 0.07 mmHg) and mackerel (1.39 +/- 0.03 mmHg). Because the oxygen affinity of Mb decreases with increasing temperature, the above differences in oxygen affinity between endothermic and ectothermic fish are reduced when adjusted for the in vivo muscle temperature of the animal. Oxygen dissociation rate constants at 20 degrees C for the endothermic species ranged from 34.1 to 49.3 s(-1), whereas those for mackerel and bonito were 102 and 62 s(-1), respectively. Correlated with the low oxygen affinity and fast dissociation kinetics of mackerel Mb is a substitution of alanine for proline that would likely result in a more flexible mackerel protein.

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin.

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.

Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket.

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe-protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s(-1), is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6-coordinate and low-spin in both ferric and ferrous forms in the pH range 5.5-10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino-acid sequence comparison, is involved in the formation of the ferric and ferrous 6-coordinate low-spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5-coordinate and high-spin and the ferric form contains a 6-coordinate high-spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.

Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: evidence for ligation of tyrosine-63 (B10) to the heme.

We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single-amino acid mutants by EPR, optical absorbance, and resonance Raman spectroscopy. The helical positions of glutamine-84, tyrosine-63, and lysine-87 are suggested to correspond to E7, B10, and E10, respectively, in the distal heme pocket on the basis of amino acid sequence comparison of mammalian globins. The protein undergoes a transition with a pK of 6.3 from a six-coordinate high-spin aquomet form at acidic pH to a six-coordinate low-spin form. The EPR signal of the low-spin form for the wild-type protein is absent for the Tyr63Leu mutant, suggesting that the B10 tyrosine in the wild-type protein ligates to the heme as tyrosinate. For the Tyr63Leu mutant, a new low-spin signal resembling that of alkaline cytochrome c (a His-heme-Lys species) is resolved, suggesting that the E10 lysine now coordinates to the heme. In the wild-type protein, the oxygen of the tyrosine-63 side chain is likely to share a proton with the side chain of lysine-87, suggested by the observation of a H/D sensitive resonance Raman line at 502 cm(-)(1) that is tentatively assigned as a vibrational mode of the Fe-O bond between the iron and the tyrosinate. We propose that the transition from the high-spin to the low-spin form of the protein occurs by deprotonation and ligation to the heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.

A cooperative oxygen-binding hemoglobin from Mycobacterium tuberculosis.

Two putative hemoglobin genes, glbN and glbO, were recently discovered in the complete genome sequence of Mycobacterium tuberculosis H37Rv. Here, we show that the glbN gene encodes a dimeric hemoglobin (HbN) that binds oxygen cooperatively with very high affinity (P(50) = 0.013 mmHg at 20 degrees C) because of a fast combination (25 microM(-1).s(-1)) and a slow dissociation (0.2 s(-1)) rate. Resonance Raman spectroscopy and ligand association/dissociation kinetic measurements, along with mutagenesis studies, reveal that the stabilization of the bound oxygen is achieved through a tyrosine at the B10 position in the distal pocket of the heme with a conformation that is unique among the globins. Physiological studies performed with Mycobacterium bovis bacillus Calmette-Guérin demonstrate that the expression of HbN is greatly enhanced during the stationary phase in aerobic cultures but not under conditions of limited oxygen availability. The results suggest that, physiologically, the primary role of HbN may be to protect the bacilli against reactive nitrogen species produced by the host macrophage.

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.

Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands.

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas 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 (>10(7) microM-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 heme environment in barley hemoglobin.

To elucidate the environment and ligand structure of the heme in barley hemoglobin (Hb), resonance Raman and electron paramagnetic resonance spectroscopic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Barley Hb has a unique heme environment as judged from the Fe-CO and C-O stretching frequencies in the CO complex. Two Fe-CO stretching modes are observed with frequencies at 534 and 493 cm-1, with relative intensities that are pH sensitive. The 534 cm-1 conformer shows a deuterium shift, indicating that the iron-bound CO is hydrogen-bonded, presumably to the distal histidine. A C-O stretching mode at 1924 cm-1 is assigned as being associated with the 534 cm-1 conformer. Evidence is presented that the high Fe-CO and low C-O stretching frequencies (534 and 1924 cm-1, respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm-1 conformer arises from an open conformation of the heme pocket and becomes the dominant population under acidic conditions when the distal histidine moves away from the CO. Strong hydrogen bonding between the bound ligand and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stability to the bound oxygen. This stabilization is confirmed by the dramatic decrease in the oxygen dissociation rate compared with sperm whale myoglobin.

Solution and crystal structures of a sperm whale myoglobin triple mutant that mimics the sulfide-binding hemoglobin from Lucina pectinata.

The bivalve mollusc Lucina pectinata harbors sulfide-oxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O2, but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86-89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with approximately 700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln64(E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe68(E11) is rotated approximately 90 degrees about chi2 and located approximately 1-2 A closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.

A comparison of functional and structural consequences of the tyrosine B10 and glutamine E7 motifs in two invertebrate hemoglobins (Ascaris suum and Lucina pectinata).

The architecture of the distal heme pocket in hemoglobins and myoglobins can play an important role in controlling ligand binding dynamics. The size and polarity of the residues occupying the distal pocket may contribute steric and dielectric effects. In vertebrate systems, the distal pocket typically contains a "distal" histidine at position E7 and a leucine at position B10. There are several invertebrate organisms that have hemoglobins or myoglobins that display a pattern in which residues E7 and B10 are a glutamine and tyrosine, respectively. These proteins often have very high oxygen affinities stemming from very slow ligand off rates. In this study, two such hemoglobins, one from the nematode Ascaris suum and the other from the sulfide-fixing clam Lucina pectinata, are compared with respect to conformational and functional properties. Ultraviolet resonance Raman spectroscopy and visible resonance Raman spectroscopy are used to probe, respectively, the ligand-dependent hydrogen bonding pattern of the tyrosine residues and the proximal heme pocket interactions. Fourier transform infrared absorption spectroscopy is used to probe the dielectric properties of the distal heme pocket through the stretching frequency of carbon monoxide bound to the heme. Functionality is probed through the geminate rebinding of both CO and O2. The findings reveal two very different patterns indicative of two different mechanisms for achieving low oxygen off rates. In Hb Ascaris, a hydrogen bonding network that includes the E7 Gln, B10 Tyr, and oxygen bound to the heme results in a tight cage for the oxygen. Dissociation of the O2 requires a large amplitude conformational fluctuation that results both in a spontaneous dissociation of the oxygen through the loss of hydrogen bond stabilization and in an enhanced probability for ligand escape though the transient disruption and opening of the tight distal cage. In the case of the Hb from Lucina, there is no evidence for a tight cage. Instead the data support a model in which the hydrogen bonding network is far more tenuous and the equilibrium state of distal pocket is far more open and accessible than is the case in Ascaris. The results explain why Hb Ascaris has one of the highest oxygen affinities known (P50 approximately 10(-)3 Torr) while Hb Lucina II has an oxygen affinity comparable to that of Mb (P50 = 0.13 Torr) even though both of these Hbs contain the B10 Tyr and E7 Gln motif and display very low oxygen off rates. The roles of water and proximal strain are discussed.

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.

Circular dichroism spectroscopy of Lucina I hemoglobin.

The monomeric hemoglobin from the mollusc Lucina pectinata (HbI) represents an interesting model system for the study of heme-related circular dichroic (CD) bands in view of the highly asymmetric distribution of aromatic residues around the heme pocket revealed by the X-ray crystal structure. The CD spectra of both ferrous and ferric HbI derivatives exhibit negative CD bands in the Soret and ultraviolet region with an enhanced ellipticity of the heme N and L bands in the near-UV region. In contrast, the magnitude of the Cotton effect in the visible and Soret regions is comparable to that observed in other hemoproteins. The spectrum of the carbon monoxide derivative shows a surprising similarity with that observed for the soybean leghemoglobin carbon monoxide adduct. A common structural feature in the two proteins is the presence in the distal pocket of two Phe residues (B9 and B10) the aromatic rings of which are perpendicular to the heme plane.

Nonsteric factors dominate binding of nitric oxide, azide, imidazole, cyanide, and fluoride to the rhizobial heme-based oxygen sensor FixL.

BACKGROUND: The FixL protein is a heme-based sensor. Binding of oxygen to a unique heme domain inhibits a kinase domain of the type found in two-component regulators. Oxygen association is slow, but the dissociation rate is comparable to that of myoglobins. We have probed the size and chemistry of the FixL heme pocket by measuring the affinites, on rates and off rates for a wide variety of ferric heme ligands. Cyanide, but not fluoride, regulates the kinase activity. To examine how the sensory heme domain interacts with the kinase, we asked how the presence of the kinase domain affects ligand binding. RESULTS: The affinities of ferric FixL for heme ligands follow the same trend as their pKa values: cyanide > 4-methyl imidazole > imidazole > fluoride > azide >> thiocyanate. The association rates follow the reverse trend. Striking differences from myoglobin include a 6-fold greater affinity for, and faster binding to, the bulky ligand imidazole, a 14-fold faster on rate for nitric oxide, a 2 800-fold lower affinity for azide, and a complete failure to bind thiocyanate. The presence of the kinase domain does not alter the affinity or binding kinetics of the high-spin ligand fluoride, but affects the off rates of other ligands. The EPR spectrum shows a characteristic pentacoordinate nitrosyl heme, indicating that the Fe-His bond in FixL is strained. CONCLUSIONS: The importance of ligand deprotonation to the on rates and the fact that large ligands bind readily indicate that the heme pocket is open and apolar. Ligand basicity strongly influences the strength of binding. The destabilization of inhibitory ligands by the presence of the kinase domain is consistent with a 'load' imposed by coupling to the inactivating mechanism.

Structural bases for sulfide recognition in Lucina pectinata hemoglobin I.

The X-ray crystal structure of the sulfide derivative of ferric Lucina pectinata hemoglobin component I (HbI) has been determined at 1.9 A resolution (R-factor 0.186). The heme pocket structural organization of HbI is in keeping with its ligand binding properties. The fast sulfide association rate constant can be related to the presence of Gln(64)E7, as the heme distal residue, together with the protein structural properties in the CD-E distal region. Moreover, the very high sulfide affinity for HbI is reflected by the exceptionally slow ligand dissociation rate. The stabilization of the heme-bound sulfide molecule is achieved through hydrogen bonding to Gln(64)E7, as well as by finely tuned aromatic-electrostatic interactions with the clustered residues Phe(29)B10, Phe(43)CD1 and Phe(68)E11. Such a peculiar arrangement of phenylalanyl residues at the distal ligand binding site has not been observed before in the globin family, and is unique to HbI, a protein functionally devoted to sulfide transport.

Hemoglobin in the symbiont-harboring gill of the marine gastropod Alviniconcha hessleri.

Hydrogen sulfide of geochemical origin, mixing at oceanic hydrothermal vents with oxygen from oceanic seawater, supports dense populations of chemoautrophic, sulfur-oxidizing bacteria. Those animals, the vestimentiferan worm Riftia pachyptila, certain bivalve molluscs, and the recently discovered Pacific gastropod Alviniconcha hessleri, that interiorize the bacteria as intracellular symbionts dominate the vent fauna (1, 2). The immense size of these animals, the large standing crop represented in their dense communities, and the rapid growth of individuals all attest to the effective use of an abundant food base. Dense concentrations of the mesogastropod Alviniconcha hessleri (2, 3) were recently discovered at deep-sea hydrothermal vents at the spreading center in the Mariana Back-Arc Basin of the Western Pacific. These animals house chemoautrophic, sulfide-oxidizing bacteria within specialized cells (bacteriocytes) of their modified gills (2). They are the only reported example of a symbiotic association between a gastropod mollusc and intracellular chemoautotrophic bacteria. We now show that the modified gill of Alviniconcha contains hemoglobin at a concentration of about 65 mumol/kg wet weight gill. This value falls within the range, 20-250 mumol hemoglobin per kilogram, encountered in the modified symbiont-harboring gills of many of the sulfide-dependent clams examined but is short of the very high concentrations, 550 and 1200 mumol/kg, found in Myrtea spinifera and Lucina pectinata respectively (4). Accordingly, bacteriocyte hemoglobin is a feature common to both gastropod and bivalve symbioses.

Structure of the sulfide-reactive hemoglobin from the clam Lucina pectinata. Crystallographic analysis at 1.5 A resolution.

The crystal structure of the aquo-met form of the sulfide-reactive hemoglobin (component I) from the gill of the symbiont-harboring mollusc, Lucina pectinata, has been solved and refined at 1.5 A resolution, based on synchrotron radiation X-ray diffraction data, and employing molecular replacement techniques. The crystallographic R-factor, calculated for the data in the 15.0 to 1.5 A resolution range, is 0.170, with highly regular stereochemical parameters for the protein model, and including 131 water molecules. The monomeric hemoglobin I chain consists of 142 amino acid residues, which have been partly identified on the basis of the crystallographic analysis. The molecule is characterized by an unusual distribution of aromatic residues, particularly in the region surrounding the distal site in the heme pocket. The heme distal residue is Gln(64)E7, while other notable amino acid substitutions include Trp(21)B2, Phe(29)B10, Leu(46)CD3, Phe(68)E11 and Trp(75)E18. An amino acid insertion (Ser44) is observed between sites CD1 and CD2. In the aquo-met protein, a water molecule is present at the sixth coordination position of the heme iron, and hydrogen bonded to Gln(64)E7. Simple model building shows that a dioxygen molecule, bound to ferrous protein, would contact with its free atom the ring edge of Phe(29)B10, being thus stabilized at the coordination site by an aromatic-electrostatic interaction. Similarly, the unique packing and organization of aromatic residues in the surroundings of the heme distal site is proposed as the molecular basis of the very high affinity of Lucina pectinata hemoglobin I for hydrogen sulfide, considered as one of the two physiological ligands of the protein.

Crystallization of hemoglobins II and III of the symbiont-harboring clam Lucina pectinata.

Diffraction data to 2.7 A resolution were measured on crystals of the homotetramers of components II and III of the cytoplasmic hemoglobin of the symbiont-harboring clam Lucina pectinata. Even though the crystallization conditions are different and the sequence homology of the two hemoglobins is only 63%, the crystals are isomorphous to each other and to the heterotetramer Hb II/III, implying that the residues primarily involved in the intermolecular interactions and responsible for crystal cohesion may be invariant.

Effects of carbon monoxide on isolated heart muscle cells.

By sequestering intracellular myoglobin of cardiac muscle cells in the nonfunctioning carboxymyoglobin form, carbon monoxide blocks myoglobin-facilitated diffusion of oxygen, as well as myoglobin-mediated oxidative phosphorylation. Here, we explore the hypothesis that the carbon monoxide blockade of myoglobin function may be responsible at the cellular level for a component of the cardiotoxicity of carbon monoxide observed during exercise. Suspensions of isolated rat cardiac myocytes were held in near steady states of oxygen pressure near the intracellular partial pressure of oxygen of the working heart (2 to 5 torr) and near the end-venous partial pressure of oxygen (20 torr). These suspensions were exposed to CO at low pressure (0.07 to 70 torr; 90 to 90,000 parts per million). The fraction of intracellular carboxymyoglobin, determined spectrophotometrically, was in good agreement with the fraction predicted from the ratio of carbon monoxide partial pressure to oxygen partial pressure. The effects observed were related to the fraction of intracellular myoglobin bound to CO. At physiological oxygen pressures no greater than 5 torr, after sequestration of approximately 50% of the myoglobin, steady-state oxygen uptake decreased significantly and was significantly less than the respiration of cell groups for which the fraction of carboxymyoglobin was 0% to 40%. When respiration is diminished, the rate of aerobic adenosine triphosphate synthesis (oxidative phosphorylation) also decreases. As in the whole heart, cytoplasmic adenosine triphosphate concentration in isolated heart cells is controlled at a constant level by the creatine phosphokinase equilibrium. When adenosine triphosphate utilization is unchanged, a sensitive monitor of the decreased adenosine triphosphate synthesis is the ratio of phophocreatine to adenosine triphosphate. When carboxymyoglobin was at least 40% of the total intracellular myoglobin, we found that the ratio of phosphocreatine to adenosine triphosphate in carbon monoxide-treated heart cells was significantly lower than that in control cells from the same preparation. Thus, we concluded that sequestering intracellular myoglobin as carboxymyoglobin significantly decreased the rate of oxidative phosphorylation of isolated cardiac myocytes. We estimate that intracellular myoglobin-dependent oxidative phosphorylation will be inhibited when approximately 20% to 40% of the arterial hemoglobin in the whole animal is carboxyhemoglobin.

Role of hydrogen bonding to bound dioxygen in soybean leghemoglobin.

Electron spin echo envelope modulation (ESEEM) spectroscopy was applied to oxy cobaltous soybean leghemoglobin (oxyCoLb) in D2O at various pH values to investigate electron nuclear superhyperfine coupling to N epsilon of the proximal histidyl imidazole and to exchangeable deuterons. Two spectroscopically distinct forms of oxyCoLb, acid and neutral, were identified. In the acid form, a 0.82-MHz hyperfine coupling to 2H was found, indicating the presence of a hydrogen bond to bound O2. No hyperfine-coupled 2H was found in the neutral form. Nuclear hyperfine and nuclear quadrupole couplings to the proximal histidyl N epsilon in the acid form are smaller than those in the neutral form: Aiso = 2.22 MHz and e2qQ = 1.98 MHz for the acid form; Aiso = 2.90 MHz and e2qQ = 2.22 MHz for the neutral form. The differences are believed to result from the presence of a hydrogen bond to bound O2 in the acid form. A discussion of the contribution of this hydrogen bond to the pH-dependent O2 affinity of leghemoglobin is presented.

The amino acid sequence of hemoglobin III from the symbiont-harboring clam Lucina pectinata.

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clam Lucina pectinata consists of 152 amino acid residues, has a calculated Mm of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66. Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnson et al., J. Prot. Chem. 10, 609-622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 A of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the two Lucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the two Lucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36-49% when compared to the two groups of mollusc globins. The highest homology appears to exist between the Lucina globins and the cytoplasmic hemoglobin of Busycon canaliculatum.