Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

Synechocystis hemoglobin contains an unprecedented covalent bond between a nonaxial histidine side chain (H117) and the heme 2-vinyl. This bond has been previously shown to stabilize the ferric protein against denaturation, and also to affect the kinetics of cyanide association. However, it is unclear why Synechocystis hemoglobin would require the additional degree of stabilization accompanying the His117-heme 2-vinyl bond because it also displays endogenous bis-histidyl axial heme coordination, which should greatly assist heme retention. Furthermore, the mechanism by which the His117-heme 2-vinyl bond affects ligand binding has not been reported, nor has any investigation of the role of this bond on the structure and function of the protein in the ferrous oxidation state. Here we report an investigation of the role of the Synechocystis hemoglobin His117-heme 2-vinyl bond on structure, heme coordination, exogenous ligand binding, and stability in both the ferrous and ferric oxidation states. Our results reveal that hexacoordinate Synechocystis hemoglobin lacking this bond is less stable in the ferrous oxidation state than the ferric, which is surprising in light of our understanding of pentacoordinate Hb stability, in which the ferric protein is always less stable. It is also demonstrated that removal of the His117-heme 2-vinyl bond increases the affinity constant for intramolecular histidine coordination in the ferric oxidation state, thus presenting greater competition for the ligand binding site and lowering the observed rate and affinity constants for exogenous ligands.     

Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.     

The dissociation of carbon monoxide from hemoglobin intermediate

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species.     

Peroxynitrite scavenging by ferrous truncated hemoglobin GlbO from Mycobacterium leprae

Mycobacterium leprae GlbO has been proposed to represent merging of both O(2) uptake/transport and scavenging of nitrogen reactive species. Peroxynitrite reacts with M. leprae GlbO(II)-NO leading to GlbO(III) via the GlbO(III)-NO species. The value of the second order rate constant for GlbO(III)-NO formation is >1x10(8)M(-1)s(-1) in the absence and presence of CO(2) (1.2x10(-3)M). The CO(2)-independent value of the first order rate constant for GlbO(III)-NO denitrosylation is (2.5+/-0.4)x10(1)s(-1). Furthermore, peroxynitrite reacts with GlbO(II)-O(2) leading to GlbO(III) via the GlbO(IV)O species. Values of the second order rate constant for GlbO(IV)O formation are (4.8+/-0.5)x10(4) and (6.3+/-0.7)x10(5)M(-1)s(-1) in the absence and presence of CO(2) (=1.2x10(-3)M), respectively. The value of the second order rate constant for the peroxynitrite-mediated GlbO(IV)O reduction (= (1.5+/-0.2)x10(4)M(-1)s(-1)) is CO(2)-independent. These data argue for a role of GlbO in the defense of M. leprae against nitrosative stress.     

Evidence of hemoglobin dissociation

Bovine carbonmonoxy hemoglobin investigated with light scattering studies is found to dissociate from its native tetramer structure into dimers and monomers. The values of the hydrated tetramer radius, R_T = 32.1 Å, and the fractional dissociation vs pH, have been obtained at different ionic strengths from the autocorrelation function of the scattered light. The results suggest that a relevant contribution to Hb dissociation is due to electrostatic effects and, by means of a model derived by Tanford, it has been possible to predict the behavior of dissociation. Among the findings of this approach, we recall the estimates of the electrostatic energy contributions to Hb dissociation, up to ? GRT, and the predicted charge of tetrameric Hb vs pH, which agrees very well with the experimental data. © 1994 John Wiley & Sons, Inc.     

Kinetic light scattering studies on the dissociation of hemoglobin from Lumbricus terrestris.

The kinetics of the pH-induced dissociation of the 3 X 10(6) mol wt hemoglobin from Lumbricus terrestris (the earthworm) have been studied in a light-scattering stopped-flow apparatus. The ligand dependent dissociation data were fit well by a simple sequential model. The data for CO and oxyhemoglobin are consistent with Hb12 leads to 2Hb6 leads to 12Hb. Methemoglobin at pH 7 appears to be hexameric and the dissociation is consistent with the model: Hb6 leads to 6Hb. In a sequential decay scheme for which light-scattering changes are monitored, the relative amounts of rapid and slow phase are determined by the rate constants as well as the molecular weights of intermediate species. Assignment of the hexameric intermediate is supported by an investigation of the sensitivity of the theoretical kinetic curves to the molecular weights of the intermediates. This assignment is further supported by the following: (1) the same model will fit the data for oxy- and CO-hemoglobin at all three temperatures (a 24-29-fold variation in rate constants), (2) evidence from electron microscopy shows hexameric forms, and (3) methemoglobin is apparently stable as a hexamer at pH 7. When CO replaces O2 as the ligand, the dissociation rate increases by a factor of four. The met is about 20 times faster than the initial oxyhemoglobin dissociation rate, but perhaps more relevant for comparing dissociation of the hexamer, the met rate was respectively 100 times and 500 times faster than that for the assumed hexameric forms of CO- and oxy-hemoglobin. The activation energies for the dodecamer to hexamer dissociation and for the dissociation of the hexamer to smaller forms were about 30 kcal/mol for oxy-, CO-, and methemoglobin.     

Autoxidation of hemoglobin enhanced by dissociation into dimers.

Autoxidation as a function of hemoglobin concentration indicates a 17-fold increase in the rate of autoxidation from 0.25 (%/h) to 4.3 (%/h) when tetrameric oxyhemoglobin dissociates into dimers. As a result of this large enhancement, a contribution of dissociation to the autoxidation is evident even at relatively high concentrations of hemoglobin for which it is usually considered that dissociation can be neglected. The mechanism for this phenomenon is attributed to alterations in the ligand pocket which occur when constraints due to subunit contacts within the R-state are eliminated.     

Functional consequences of ligand-linked dissociation in hemoglobin from the sea cucumber Molpadia arenicola.

It has been established that Molpadia hemoglobin tends to dissociate into subunits as oxygen is bound. The kinetics and equilibria of carbon monoxide and ethylisocyanide binding reported here show a dependence on protein concentration that supports the conclusion that the aggregated hemoglobin has a lower ligand affinity than the dissociated subunits. This is true for the isolated D-chain as well as for the unfractionated hemolysate that contains four distinct polypeptide chains (A-D). This indicates that even homopolymers of Molpadia hemoglobin have lower ligand affinity than the dissociated subunits. At high protein concentration hemolysates of Molpadia hemoglobin show slight cooperativity. The time course of ligand binding to the deoxy D-chain also suggests cooperative interactions. The low affinity of the aggregated state may have a different molecular explanation than in human hemoglobin where tetramers of identical subunits (as in Hb H) show high oxygen affinity. The absence of tyrosine and histidine at the C-terminal of the Molpadia D-chains also suggests a different stabilization of the low affinity deoxy state. An additional functional difference between Molpadia hemoglobin and human hemoglobin is that organic phosphates do not alter the ligand affinity of the sea cucumber hemoglobin.     

Analytical isotachophoresis in capillary tubes. Analysis of hemoglobin, hemiglobin cyanide and isoelectric fractions of hemiglobin cyanide.

The zone stabilization in capillary isotachophoresis in the water phase has been improved by methylcellulose so that proteins can be analysed. Hemoglobin and hemiglobin cyanide samples were studied as model systems. Ampholine carrier ampholytes were used as spacers, enhancing the detection of the different components. The optimal amounts of Ampholine, however, were found to be much smaller than in most of the previously published reports. Linear relationships were found between the zone lengths and sample amounts, including spacers. The separations were reproducible and reached the isotachophoretic steady state. The hemiglobin cyanide was fractionated by isoelectric focusing. The four main fractions were then analyzed by capillary isotachophoresis and shown to be heterogeneous in mobility with a pH of 7.5 in the leading electrolyte. The component zones of the total hemiglobin cyanide sample were all identified in relation to the isotachophoretic components of the isoelectric fractions. The total analysis time was in average 30-40 min. The sample amounts were about 40 mug protein in each experiment with very small Ampholine volumes, 25-100 nl 40% (w/v).     

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.     

Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k_off). Since no techniques are available to estimate k_off, we report herewith a method to determine k_off based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k_off value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 °C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)-cyanide dissociation, the k_off value, representing the rate-limiting step, can be directly determined. The k_off value obtained experimentally matches very well to that calculated from values of the second-order rate constant (k_on) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (k_off = k_on × K).     

*NO dissociation represents the rate limiting step for O2-mediated oxidation of ferrous nitrosylated Mycobacterium leprae truncated hemoglobin O

Mycobacterium leprae truncated hemoglobin O (trHbO) protects from nitrosative stress and sustains mycobacterial respiration. Here, kinetics of M. leprae trHbO(II)-NO denitrosylation and of O(2)-mediated oxidation of M. leprae trHbO(II)-NO are reported. Values of the first-order rate constant for *NO dissociation from M. leprae trHbO(II)-NO (k(off)) and of the first-order rate constant for O(2)-mediated oxidation of M. leprae trHbO(II)-NO (h) are 1.3 x 10(-4) s(-1) and 1.2 x 10(-4) s(-1), respectively. The coincidence of values of k(off) and h suggests that O(2)-mediated oxidation of M. leprae trHbO(II)-NO occurs with a reaction mechanism in which *NO, that is initially bound to heme(II), is displaced by O(2) but may stay trapped in a protein cavity(ies) close to heme(II). Next, M. leprae trHbO(II)-O(2) reacts with *NO giving the transient Fe(III)-OONO species preceding the formation of the final product M. leprae trHbO(III). *NO dissociation from heme(II)-NO represents the rate limiting step for O(2)-mediated oxidation of M. leprae trHbO(II)-NO.     

Dissociation of hemoglobin into subunits. Ligand-linked dissociation at neutral pH

The dimer-tetramer association-dissociation equilibrium of hemoglobin is strongly ligand-linked at neutral pH; the ratio of the association constant of unliganded to that of oxyhemoglobin is estimated from ultracentrifuge data to be not less than 10³ in sodium chloride solutions and probably not less than 10⁵ in sodium iodide solutions. This finding affords an explanation of the fact that the ligand binding characteristics of hemoglobin are to a first approximation independent of the degree of dissociation of oxyhemoglobin—the “salt paradox”.     

The action of hemoglobin. Cooperative effects in tetrameric proteins

The cooperative effect is developed for proteins containing four subunits. The extension of the primitive Adair model is considered and the present models based on this extension are compared.