Acceleration of tetramer formation by the binding of inositol hexaphosphate to hemoglobin dimers.

The aggregation of deoxyhemoglobin dimers was studied by dropping the pH of a dilute solution of deoxyhemoglobin originally at high pH. In the presence of inositol hexaphosphate, a sharp increase in the rate of dimer association was observed. At higher concentrations of the phosphate, the rate decreased to a value close to that seen in the absence of phosphate. These observations require that inositol hexaphosphate binds to deoxyhemoglobin dimers. The dependence of the aggregation rate on phosphate concentration occurs because the reaction of a dimer containing bound phosphate with a phosphate-free dimer is 30 to 50 times faster than either the association of phosphate-free dimers or the association of dimers both containing bound phosphate.     

Enthalpy changes for inositol hexaphosphate binding to hemoglobins A and M Iwate.

Enthalpies of inositol hexaphosphate (IHP) binding to deoxy and carbonmonoxy (CO) HbA and HbM Iwate have been determined calorimetrically and compared as functions of pH. Values for deoxy HbA and for deoxy HbM Iwate are similar with CO HbM Iwate yielding slightly less heat of reaction. The results support the existence of both deoxy and CO HbM Iwate in T-like structures with only minor modifications occurring upon CO binding. For HbA observed heats of IHP binding have been corrected for heats of extraction of reacting protons from buffer. The resulting intrinsic IHP binding enthalpies show consistent values of ?7 to ?11 kcal/mol proton absorbed in binding. We suggest that a major driving force for organic phosphate binding is the exothermic protonation of histidine and/or a α-amino nitrogens induced by proximity of phosphate negative charges.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

Equilibrium and kinetic measurements of carbon monoxide binding to hemoglobin kansas in the presence of inositol hexaphosphate

Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun., 49, 1485–1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity, as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{mm}$ Hg and K_eq = 5.4 × 10⁵M^?1) and almost no cooperativity (n = 1.1) at pH 7, 25 °C. The CO “on” and “off” kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K_eq = 5.2 × 10⁵M^?1 with k_on = 1.03 × 10⁵M^?1 · S^? and k_off = 0.198 S^?1) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.     

Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine

The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction.     

Analogous effect of protons and inositol hexaphosphate on the alteration of structure of nitrosyl fetal human hemoglobin.

We have determined the low temperature EPR spectra and room temperature ligand dissociation rate constants of human NO-hemoglobins F and A as a function of pH and inositol hexaphosphate levels in order to assess the contribution of a quaternary structural equilibrium in the two proteins to their spectral and functional properties. Our results are consistent with an increased stability of a ligated low affinity structure in the fetal protein; the functional properties of this structure appear to be essentially the same in the two hemoglobins, even though its stability relative to a high affinity conformation is different. The pH dependence of the NO dissociation constant in both adult and fetal hemoglobin can be assigned primarily to the pH-dependent equilibria of high and low affinity forms as monitored by EPR.     

An investigation of the distal histidyl hydrogen bonds in oxyhemoglobin: effects of temperature, pH, and inositol hexaphosphate

On the basis of X-ray crystal structures and electron paramagnetic resonance (EPR) measurements, it has been inferred that the O(2) binding to hemoglobin is stabilized by the hydrogen bonds between the oxygen ligands and the distal histidines. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO(2) A) in solution. Here, the NMR spectra of uniformly (15)N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH and temperature and also in the presence of an organic phosphate, inositol hexaphosphate (IHP). We have found significant effects of pH and temperature on the strength of the H-bond markers, i.e., the cross-peaks for the side chains of the two distal histidyl residues, α58His and β63His, which form H-bonds with the O(2) ligands. At lower pH and/or higher temperature, the side chains of the distal histidines appear to be more mobile, and the exchange with water molecules in the distal heme pockets is faster. These changes in the stability of the H-bonds with pH and temperature are consistent with the changes in the O(2) affinity of Hb as a function of pH and temperature and are clearly illustrated by our NMR experiments. Our NMR results have also confirmed that this H-bond in the β-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. IHP has only a minor effect on these H-bond markers compared to the effects of pH and temperature. These H-bonds are sensitive to mutations in the distal heme pockets but not affected directly by the mutations in the quaternary interfaces, i.e., α(1)β(1) and/or α(1)β(2) subunit interface. These findings provide new insights regarding the roles of temperature, hydrogen ion, and organic phosphate in modulating the structure and function of hemoglobin in solution.     

The effect of inositol hexaphosphate on the absorption spectra of alpha and beta chains in nitrosyl hemoglobin.

The spectral changes of nitrosyl hemoglobin on addition of inositol hexaphosphate were studied in hybrid-heme hemoglobins. The results showed that the decrease in absorption in the Soret region was mainly due to a spectral change in alpha chains, and that the tension on heme in the quaternary T structure was much stronger in alpha than in beta chains.     

A proton magnetic relaxation study of the interaction of isolated sheep methaemoglobins A,B and C with inositol hexaphosphate

• 1.1. Sheep aquomethaemoglobins A and B exert similar paramagnetic influence on the solventproton magnetic relaxation (PMR) rates as observed in human HbA, even in the presence of inositol hexaphosphate (IHP).
• 2.2. On the other hand, phosphate-free methaemoglobin C induces smaller PMR rates due to the restricted haem-accessibility.
• 3.3. Here IHP also binds, although with smaller affinity and with different structural consequences from the other two sheep haemoglobins. Some implications of these findings are discussed here.

     

The effects of inositol hexaphosphate on the allosteric properties of two beta-99-substituted abnormal hemoglobins, hemoglobin Yakima and hemoglobin Kempsey.

Hemoglobins (Hb) Yakima and Kempsey were purified from patients' blood with diethylaminoethyl cellulose column chromatography. The oxygen equilibrium curves of the two hemoglobins and the effects of organic phosphates on the function were investigated. In 0.1 M phosphate buffer, Hill's constants n for Hb Yakima and Hb Kempsey were 1.0 to 1.1 at the pH range for 6.5 to 8.0 and the oxygen affinities of both the mutant hemoglobins were about 15 to 20 times that of Hb A at pH 7.0. The Bohr effect was normal in Hb Yakima and one-fourth normal in Hb Kempsey. In the presence of inositol hexaphosphate, the oxygen affinities to Hb Yakima and Hb Kempsey were greatly decreased, and an interesting result revealed that these hemoglobins showed clear cooperativity in oxygen binding. Hill's constant n in the presence of inositol hexaphosphate was 1.9 for Hb Kempsey and 2.3 for Hb Yakima at pH 7.0. The cooperativities of these mutant hemoglobins were pH-dependent, and Hb Kempsey showed high cooperativity at low pH (n equal 2.1 at pH 6.6) and low cooperativity at high pH (n equal 1.0 at pH 8.0). Hb Yakima showed similar pH dependence in cooperativity. In the presence of inositol hexaphosphate, Hb A showed a pH-dependent cooperativity different from those of Hb Yakima and Hb Kempsey, namely, Hill's n was the highest in alkaline pH (n equal 3.0 at pH 8.0) and decreased at lower pH (n equal 1.5 at pH 6.5). 2,3Diphosphoglycerate bound with the deoxygenated Hb Yakima and Hb Kempsey, however, had no effect on the oxygen binding of these abnormal hemoglobin. The pH-dependent cooperativity of alpha1beta2 contact anomalous hemoglobin and normal hemoglobin was explained by the shifts in the equilibrium between the high and low ligand affinity forms.     

Substitution of the heme binding module in hemoglobin alpha- and beta-subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin

In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)H proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.     

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.     

Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.     

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.     

A nuclear Overhauser effect study of the heme crevice in the resting state and compound I of horseradish peroxidase: evidence for cation radical delocalization to the proximal histidine

The assignment of resolved hyperfine-shifted resonances in high-spin resting state horseradish peroxidase (HRP) and its double-oxidized reactive form, compound I (HRP-I), has been carried out by using the nuclear Overhauser effect (NOE) starting with the known heme methyl assignments in each species. In spite of the efficient spin-lattice relaxation and very broad resonances, significant NOEs were observed for all neighboring pyrrole substituents, which allowed the assignment of the elusive propionate alpha-methylene protons. In the resting state HRP, this leads directly to the identity of the proximal His-170 H beta peaks. The determination that one of the most strongly contact-shifted single proton resonances in HRP-I does not arise from the porphyrin dictates that the cation radical must be delocalized to some amino acid residue. The relaxation properties of the non-heme contact-shifted signal in HRP-I support the identity of this contributing residue as the proximal His-170. Detailed analysis of changes in both contact shift pattern and NOEs indicates that compound I formation is accompanied by a approximately 5 degree rotation of the 6-propionate group. The implication of a porphyrin cation radical delocalized over the proximal histidine for the proposed location of the solely amino acid centered radical in compound I of related cytochrome c peroxidase is discussed.     

Restricting the ligand-linked heme movement in Scapharca dimeric hemoglobin reveals tight coupling between distal and proximal contributions to cooperativity.

Cooperative ligand binding in the dimeric hemoglobin from the blood clam Scapharca inaequivalvis results primarily from tertiary, rather than quaternary, structural changes. Ligand binding is coupled with conformational changes of key residues, including Phe 97, which is extruded from the proximal heme pocket, and the heme group, which moves deeper into the heme pocket. We have tested the role of the heme movement in cooperative function by mutating Ile 114, at the base of the heme pocket. Replacement of this residue with a Met did not disturb the hemoglobin structure or significantly alter equilibrium ligand binding properties. In contrast, substitution with a Phe at position 114 inhibits the ligand-linked movement of the heme group, and substantially reduces oxygen affinity and cooperativity. As the extent of heme movement to the normal position of the ligated state is diminished, Phe 97 is inhibited from its movement into the interface upon ligand binding. These results indicate a tight coupling between these two key cooperative transitions and suggest that the heme movement may be an obligatory trigger for expulsion of Phe 97 from the heme pocket.