Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Human haemoglobin: a new paradigm for oxygen binding involving two types of alphabeta contacts.

This review summarizes the most recent state of haemoglobin (Hb) research based on the literature and our own results. In particular, an attempt is made to form a unified picture for haemoglobin function by reconciling the cooperative oxygen binding with the stabilization of the bound dioxygen in aqueous solvent. The HbA molecule contains two types of alphabeta contacts. One type is the alpha1beta2 or alpha2beta1 contacts, called sliding contacts, and these are strongly associated with the cooperative binding of O2 to the alpha2beta2 tetramer. The other type is the alpha1beta1 or alpha2beta2 contacts, called packing contacts, but whose role in Hb function was not clear until quite recently. However, detailed pH-dependence studies of the autoxidation rate of HbO2 have revealed that the alpha1beta1 and alpha2beta2 interfaces are used for controlling the stability of the bound O2. When the alpha1beta1 or alpha2beta2 contact is formed, the beta chain is subjected to a conformational constraint which causes the distal (E7) histidine to be tilted slightly away from the bound dioxygen, preventing the proton-catalysed nucleophilic displacement of O2- from the FeO2 by an entering water molecule. This is one of the most characteristic features of HbO2 stability. Finally we discuss the role of the alpha1beta1 or alpha2beta2 contacts by providing some examples of unstable haemoglobin mutants. These pathological mutations are found mostly on the beta chain, especially in the alpha1beta1 contact regions. In this way, HbA seems to differentiate two types of alphabeta contacts for its functional properties.     

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.     

Conformational substates of the oxyheme centers in alpha and beta subunits of hemoglobin as disclosed by EPR and ENDOR studies of cryoreduced protein

Exposure of frozen solutions of oxyhemoglobin to gamma-irradiation at 77 K yields EPR- and ENDOR-active, one-electron-reduced oxyheme centers which retain the conformation of the diamagnetic precursor. EPR spectra have been collected for the centers produced in human HbO(2) and isolated alphaO(2) and betaO(2) chains, as well as alphaO(2)beta(Zn), alpha(Zn)betaO(2), and alphaO(2)beta(Fe(3+)) hybrids, each in frozen buffer and in frozen glasses that form in the presence of glycols and sugars and also in the presence of IHP. These reveal two spectroscopically distinct classes of such ferriheme centers (g(1) 相似文献   

A flow procedure to determine oxygen binding isotherms for low affinity and easily oxidized hemoglobins.

1. The determination of binding isotherms for low affinity hemoglobins is particularly difficult because of rapid autoxidation. 2. In carp Hb (pH 6 + IHP, 25 degrees C), one-quarter of the hemes are oxidized within 3 min, preventing the accurate determination of even P50 or X. 3. We circumvent this problem by rapidly flowing HbO2, initially at pH 9 (X = 1.4 microM), against a low pH buffer to bring the system rapidly to equilibrium in the low affinity form. Diode-array spectrophotometry allows a complete spectrum to be obtained less than 5 sec, after flow ceases, before significant oxidation has occurred. In tandem with the stopped flow apparatus is an oxygen electrode to measure O2 activity. 4. At 22 degrees C, the half-saturation oxygen activity (X) is 227 microM, and the Hill number is 0.91, for carp Hb (T state) implying significant differences in the O2 affinities of the alpha and beta chain hemes or, two different T states.     

Spectral changes upon subunit association in valency hybrid hemoglobins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact.     

Nature of the FeO2 bonding in myoglobin and hemoglobin: A new molecular paradigm

The iron(II)-dioxygen bond in myoglobin and hemoglobin is a subject of wide interest. Studies range from examinations of physical-chemical properties dependent on its electronic structure, to investigations of the stability as a function of oxygen supply. Among these, stability properties are of particular importance in vivo. Like all known dioxygen carriers synthesized so far with transition metals, the oxygenated forms of myoglobin and hemoglobin are known to be oxidized easily to their ferric met-forms, which cannot bind molecular oxygen and are therefore physiologically inactive. The mechanistic details of this autoxidation reaction, which are of clinical, as well as of physical-chemical, interest, have long been investigated by a number of authors, but a full understanding of the heme oxidation has not been reached so far. Recent kinetic and thermodynamic studies of the stability of oxymyoglobin (MbO2) and oxyhemoglobin (HbO2) have revealed new features in the 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 or HbO2 in the form of O2- so that the iron is converted to the ferric met-form. Since the autoxidation reaction of MbO2 or HbO2 proceeds through a nucleophilic displacement following one-electron transfer from iron(II) to the bound O2, this reaction may be viewed as a meeting point of the stabilization and the activation of molecular oxygen performed by hemoproteins. Along with these lines of evidence, we finally discuss the stability property of human HbO2 and provide with the most recent state of hemoglobin research. The HbA molecule contains two types of alphabeta contacts and seems to differentiate them quite properly for its functional properties. The alpha1beta2 or alpha2beta1 contact is associated with the cooperative oxygen binding, whereas the alpha1beta1 or alpha2beta2 contact is used for controlling the stability of the bound O2. We can thus form a unified picture for hemoglobin function by closely integrating the cooperative and the stable binding of molecular oxygen with iron(II) in aqueous solvent. These new views on the nature of FeO2 bonding and the possible role of globin moiety in stabilizing MbO2 and HbO2 are of primary importance, not only for a full understanding of various hemoprotein reactions with O2, but also for planning new molecular designs for synthetic oxygen carriers which may be able to function in aqueous solvent and at physiological temperature.     

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.     

The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins.

Some insects have a globin exclusively in their fast-growing larval stage. This is the case in the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in Japan. In the polymorphic hemoglobin comprised of 11 separable components, hemoglobin VII (Ta-VII Hb) was of particular interest. When its ferric met-form was exposed to pH 5.0 from 7.2, the distal histidine was found to swing away from the E7 position. As a result, the iron(III) was converted from a hexacoordinate to a pentacoordinate form by a concomitant loss of the axial water ligand. The corresponding spectral changes in the Soret band were therefore followed by stopped-flow and rapid-scan techniques, and the observed first-order rate constants of k(out) = 25 s(-1) and kin = 128 s(-1) were obtained for the outward and inward movements, respectively, of the distal histidine residue in 0.1 m buffer at 25 degrees C. For O2 affinity, Ta-VII Hb showed a value of P50 = 1.7 Torr at pH 7.4, accompanied with a remarkable Bohr effect (deltaH+ = -0.58) almost equal to that of mammalian hemoglobins. We have also investigated the stability property of Ta-VII HbO2 in terms of the autoxidation rate over a wide range of pH from 4 to 11. The resulting pH-dependence curve was compared with those of another component Ta-V HbO2 and sperm whale MbO2, and described based on a nucleophilic displacement mechanism. In light of the O2 binding affinity, Bohr effect and considerable stability of the bound O2 against acidic autoxidation, we conclude that T. akamusi Hb VII can play an important role in O2 transport and storage as the major component in the larval hemolymph.     

Aggregation of deoxyhemoglobin subunits.

The formation of deoxyhemoglobin was examined by measuring the heme spectral change that accompanies the aggregation of isolated alpha and beta chains. At low hemeconcentrations (less than 10(-5) M), tetramer formation can be described by two consecutive, second order reactions representing the aggregation of monomers followed by the association of alphabeta dimers. At neutral pH, the rates of monomer and dimer aggregation are roughly the same, approximately 5 X 10(5) M(-1) X(-1) at 20 degrees. Raising or lowering the pH results in a uniform decrease of both aggregation rates due presumably to repulsion of positively charged subunits at acid pH and repulsion of negatively charged subunits at alkaline pH. Addition of p-hydroxymercuribenzoate to alpha chains lowers the rate of monomer aggregation whereas addition of mercurials to the beta subunits appears to lower both the rate of monomer and the rate of dimer aggregation. At high heme concentrations (greater than 10(-5) M) or in the presence of organic phosphates, the rate of chain aggregation becomes limited, in part, by the slow dissociation of beta chain tetramers. In the case of inositol hexaphosphate, the rate of hemoglobin formation exhibits a bell-shaped dependence on phosphate concentration. When intermediate concentrations of inositol hexaphosphate (approximately 10(-4 M) are preincubated with beta subunits, a slow first order time course is observed and exhibits a half-time of about 8 min. As more inositol hexaphosphate is added, the chain aggregation reaction begins to occur more rapidly. Eventually at about 10(-2) M inositol hexaphospate, the time course becomes almost identical to that observed in the absence of phosphates. The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to beta monomers and, if added in sufficiently large amounts, promotes beta4 dissociation. A quantitative analysis of these results showed that the affinity of beta monomers for inositol hexaphosphate is the same as that of alphabeta dimers. Only when tetramers are formed, either alpha2beta2 or beta4, is a marked increase in affinity for inositol hexaphosphate observed.     

Autoxidation of the site-specifically PEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the autoxidation

The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.     

Characteristics of brain Kv1 channels tailored to mimic native counterparts by tandem linkage of alpha subunits: implications for K+ channelopathies

Most neuronal Kv1 channels contain Kv1.1, Kv1.2 alpha, and Kvbeta2.1 subunits, yet the influences of their stoichiometries on properties of the (alpha)(4)(beta)(4) variants remain undefined. cDNAs were engineered to contain 0, 1, 2, or 4 copies of Kv1.1 with the requisite number of Kv1.2 and co-expressed in mammalian cells with Kvbeta2.1 to achieve "native-like" hetero-oligomers. The monomeric (Kv1.1 or 1.2), dimeric (Kv1.1-1.2 or 1.2-1.2), and tetrameric (Kv1.1-(1.2)(3)) constructs produced proteins of M(r) approximately 62,000, 120,000, and 240,000, which assembled into (alpha)(4)(beta)(4) complexes. Each alpha cRNA yielded a distinct K(+) current in oocytes, with voltage dependence of activation being shifted negatively as the Kv1.1 content in tetramers was increased. Channels containing 1, 2, or 4 copies of Kv1.1 were blocked by dendrotoxin k (DTX)(k) with similarly high potencies, whereas Kv(1.2)(4) proved nonsusceptible. Accordingly, Kv1.2/beta2.1 expressed in baby hamster kidney cells failed to bind DTX(k); in contrast, oligomers containing only one Kv1.1 subunit in a tetramer exhibited high affinity, with additional copies causing modest increases. Thus, one Kv1.1 subunit largely confers high affinity for DTX(k), whereas channel electrophysiological properties are tailored by the content of Kv1.1 relative to Kv1.2. This notable advance could explain the diversity of symptoms of human episodic ataxia I, which is often accompanied by myokymia, due to mutated Kv1.1 being assembled in different combinations with wild-type and Kv1.2.     

Oxygen equilibria of hybrid-heme hemoglobins containing proto- and mesoheme groups. On the nonequivalence of alpha and beta chains.

Hybrid-heme hemoglobins, alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2, were prepared, and the O2 equilibria of their alpha and beta chains were measured separately at the isosbestic points of the partner chains at different pH values and in the presence and absence of inositol hexaphosphate. The Adair equation was extended to distinguish between the O2 saturations of the alpha and beta chains, and the seven equilibrium parameters were obtained by curve fitting to those equations. The results showed that the beta chains have an affinity slightly higher than the alpha chains in the binding of the first O2 molecule. For the second O2 molecule, the molecular species that has been oxygenated on the alpha chain has a higher affinity than that carrying O2 on the beta chain. The slopes of the Hill plots were higher for the alpha chain. The O2 saturation curves for the alpha and beta chains were calculated from the parameters averaged for the hybrids alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2 in order to cancel the effects of the heme replacement. The curves showed that the difference in O2 saturation between the two kinds of chains depends on the conditions and on the degree of O2 saturation. It was concluded that the functional difference between the chains is small enough so that it is not required to modify the models already accepted for the cooperativity of hemoglobin.     

Kinetic studies on partially liganded species of carboxyhemoglobin: alpha 2 CO beta 2 and alpha 2 beta 2CO

The valency hybrids of Hb A, alpha 2CO beta 2+, and alpha 2+ beta 2CO have been prepared by a new high pressure liquid chromatography method, and the kinetics of their CO-combination and dissociation reactions have been studied by double mixing and microperoxidase methods. Both reactions are biphasic. The slow phase in CO-combination and the fast phase in CO-dissociation are due to the reactions of alpha CO2 beta T2 or alpha 2 beta 2CO,T. The fast phase in CO-combination reaction has two components, one due to the dimers of the hybrid and the other due to the R-state tetramer. Immediately after the reduction of the valency hybrids, the overall system is represented by the equation: 2 alpha CO beta in equilibrium alpha 2CO beta 2R in equilibrium alpha 2CO beta 2T or (formula: see text) If the solutions are aged for 3-11 s, the R-state population is reduced gradually to a very small size, and the main species after 11 s of aging are dimers and T-state tetramers. Analysis of the kinetic data indicates slow R in equilibrium T equilibria in the absence of phosphates and significant dissociation of the T-state tetramer. It is concluded that the subunit contacts alpha 1-beta 2 (or alpha 2-beta 1) are impaired seriously in the hybrids. Very slow R in equilibrium T relaxation makes these hybrids unlikely intermediates in the sequential binding of CO to Hb tetramer.     

Optical and ESR-spectroscopic study of electronic adducts of oxymyoglobin and oxyhemoglobin

It has been shown that low temperatures (77 degrees K) irradiation of frozen water-glycerol solutions of oxymyoglobin and oxyhemoglobin induces kinetically stabilized nonequilibrium electronic adducts (MbO2-, HbO2-) at the expense of binding of thermolyzed electrons formed during matrix radiolysis to oxygenated hem iron. The absorption spectra of HbO2-and MbO2- have a wide band with the maximum at 545 nm and Soret's band at 421 nm. At 77 K MbO2- gives the ESR spectrum with g beta 1 = 2.203 and g beta 2 = 2.103. Unlike the latter HbO2- ESR spectrum consists of two signals g beta 1 = 2.234, g beta 2 = 2.135 and g alpha 1 = 2.195, g alpha 2 = 2.103. Two signals in HbO2- spectra are shown to be conditioned by electronic adducts of oxygenated alpha- and beta-subunits. The observed effect points to non-equivalency of O2 in alpha- and beta-subunits of oxyhemoglobin. Binding of inositolhexaphopshate to oxyhemoglobin induces changes in the electron structure of HbO2-active centres.     

Reaction pathways in the decomposition of hydrogen peroxide catalyzed by copper(II)

The kinetics of the decomposition of H(2)O(2) catalyzed by Cu(II) has been studied by the initial-rate method in aqueous phosphate media at near physiological pH. The activity of the catalyst is increased by [Fe(CN)(6)](3-) and decreased by VO(3)(-), CrO(4)(2-) and Zn(II). Three reaction pathways are involved in the Cu(II)-H(2)O(2) reaction, the kinetic orders of the catalyst being 1 (rate constant k1), 2 (rate constant k2) and 3 (rate constant k3). The three pathways present fractional apparent orders (>1) in H(2)O(2) and base catalysis. The apparent activation energies associated to rate constants k1, k2 and k3 are 102+/-4, 65+/-8 and 61+/-5 kJ mol(-1). Free-radical chain mechanisms are proposed for the three pathways.     

Characterization of functional heme domains from soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.     

Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.     

The contribution of the asymmetric alpha 1beta 1 half-oxygenated intermediate to human hemoglobin cooperativity.

Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O(2)] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O(2))beta(Fe-O(2))][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O(2) binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O(2) equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O(2)-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)]( 2) and [alpha(Zn)beta(Fe)]( 2) were measured by using an Imai apparatus. It was shown that the first and second O(2) equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg(-1), respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O(2) binding study.     

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.