Crystal structures of manganese- and cobalt-substituted myoglobin in complex with NO and nitrite reveal unusual ligand conformations

Nitrite is now recognized as a storage pool of bioactive nitric oxide (NO). Hemoglobin (Hb) and myoglobin (Mb) convert, under certain conditions, nitrite to NO. This newly discovered nitrite reductase activity of Hb and Mb provides an attractive alternative to mammalian NO synthesis from the NO synthase pathway that requires dioxygen. We recently reported the X-ray crystal structure of the nitrite adduct of ferric horse heart Mb, and showed that the nitrite ligand binds in an unprecedented O-binding (nitrito) mode to the d(5) ferric center in Mb(III)(ONO) [D.M. Copeland, A. Soares, A.H. West, G.B. Richter-Addo, J. Inorg. Biochem. 100 (2006) 1413-1425]. We also showed that the distal pocket in Mb allows for different conformations of the NO ligand (120 degrees and 144 degrees ) in Mb(II)NO depending on the mode of preparation of the compound. In this article, we report the crystal structures of the nitrite and NO adducts of manganese-substituted hh Mb (a d(4) system) and of the nitrite adduct of cobalt-substituted hh Mb (a d(6) system). We show that the distal His64 residue directs the nitrite ligand towards the rare nitrito O-binding mode in Mn(III)Mb and Co(III)Mb. We also report that the distal pocket residues allow a stabilization of an unprecendented bent MnNO moiety in Mn(II)MbNO. These crystal structural data, when combined with the data for the aquo, methanol, and azide MnMb derivatives, provide information on the role of distal pocket residues in the observed binding modes of nitrite and NO ligands to wild-type and metal-substituted Mb.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: a thermodynamic, kinetic and X-ray crystallographic study.

Thermodynamics and kinetics for cyanide, azide, thiocyanate and imidazole binding to recombinant ferric Vitreoscilla sp. homodimeric hemoglobin (Vitreoscilla Hb) have been determined at pH 6.4 and 7.0, and 20.0 degrees C, in solution and in the crystalline state. Moreover, the three-dimensional structures of the diligated thiocyanate and imidazole derivatives of recombinant ferric Vitreoscilla Hb have been determined by X-ray crystallography at 1.8 A (Rfactor=19.9%) and 2.1 A (Rfactor=23.8%) resolution, respectively. Ferric Vitreoscilla Hb displays an anticooperative ligand binding behaviour in solution. This very unusual feature can only be accounted for by assuming ligand-linked conformational changes in the monoligated species, which lead to the observed 300-fold decrease in the affinity of cyanide, azide, thiocyanate and imidazole for the monoligated ferric Vitreoscilla Hb with respect to that of the fully unligated homodimer. In the crystalline state, thermodynamics for azide and imidazole binding to ferric Vitreoscilla Hb may be described as a simple process with an overall ligand affinity for the homodimer corresponding to that for diligation in solution. These data suggest that the ligand-free homodimer, observed in the crystalline state, is constrained in a low affinity conformation whose ligand binding properties closely resemble those of the monoligated species in solution. From the kinetic viewpoint, anticooperativity is reflected by the 300-fold decrease of the second-order rate constant for cyanide and imidazole binding to the monoligated ferric Vitreoscilla Hb with respect to that for ligand association to the ligand-free homodimer in solution. On the other hand, values of the first-order rate constant for cyanide and imidazole dissociation from the diligated and monoligated derivatives of ferric Vitreoscilla Hb in solution are closely similar. As a whole, ligand binding and structural properties of ferric Vitreoscilla Hb appear to be unique among all Hbs investigated to date.     

Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: functional and antioxidative implications

The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αβ dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb-Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αβ Hb dimer. The presented results show that the Hb dimer in the Hb-Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb-Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb-Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation.     

Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.     

Intermediates detected by visible spectroscopy during the reaction of nitrite with deoxyhemoglobin: the effect of nitrite concentration and diphosphoglycerate

The reaction of nitrite with deoxyhemoglobin (deoxyHb) results in the reduction of nitrite to NO, which binds unreacted deoxyHb forming Fe(II)-nitrosylhemoglobin (Hb(II)NO). The tight binding of NO to deoxyHb is, however, inconsistent with reports implicating this reaction with hypoxic vasodilation. This dilemma is resolved by the demonstration that metastable intermediates are formed in the course of the reaction of nitrite with deoxyHb. The level of intermediates is quantitated by the excess deoxyHb consumed over the concentrations of the final products formed. The dominant intermediate has a spectrum that does not correspond to that of Hb(III)NO formed when NO reacts with methemoglobin (MetHb), but is similar to metHb resulting in the spectroscopic determinations of elevated levels of metHb. It is a delocalized species involving the heme iron, the NO, and perhaps the beta-93 thiol. The putative role for red cell reacted nitrite on vasodilation is associated with reactions involving the intermediate. (1) The intermediate is less stable with a 10-fold excess of nitrite and is not detected with a 100-fold excess of nitrite. This observation is attributed to the reaction of nitrite with the intermediate producing N2O3. (2) The release of NO quantitated by the formation of Hb(II)NO is regulated by changes in the distal heme pocket as shown by the 4.5-fold decrease in the rate constant in the presence of 2,3-diphosphoglycerate. The regulated release of NO or N2O3 as well as the formation of the S-nitroso derivative of hemoglobin, which has also been reported to be formed from the intermediates generated during nitrite reduction, should be associated with any hypoxic vasodilation attributed to the RBC.     

Interactions between flavonoids and hemoglobin in lecithin liposomes

In this paper, the binding of flavonoids (quercetin and rutin) to hemoglobin (Hb) have been investigated by fluorescence, absorption spectroscopy and circular dichroism (CD) spectroscopy. The binding parameters and binding mode between flavonoids and Hb are determined and the results of CD and synchronous fluorescence spectra indicate a conformational change of Hb with addition of flavonoids. The effects of lecithin liposomes on the binding parameter of quercetin and rutin to Hb are also studied. When incorporated into liposome, flavonoids can reduce the fluorescence of tryptophanyl residues of Hb to a lesser extent. The difference of the structure characteristics between quercetin and rutin has a significant effect on their binding affinity for Hb.     

An in silico approach to map the binding site of doxorubicin on hemoglobin

Binding modalities of doxorubicin (DOX), a widely used antineoplastic anthracyline antibiotic with hemoglobin (Hb) have been studied. The protein and the ligand were prepared using CORINA and protonated with insight II. The best conformation was sought by employing GOLDV. Molecular modeling calculations showed that DOX binds Hb to a non-classical drug binding site. The alpha subunit of Hb has been assigned to posses the binding site for DOX with a binding affinity (Ka) = 16.98 x10(3) mol(-1). The interaction was found to be thermodynamically favorable (DeltaG degrees = -66.23 KJmol(-1)). The analysis of DOX binding site to Hb suggested that the types of interactions that contribute in this binding are hydrophobic contacts, hydrogen bonding and electrostatic interactions.     

Hemoglobin S-nitrosation on oxygenation of nitrite/deoxyhemoglobin incubations is attenuated by methemoglobin

Nitrite is present in red blood cells (RBCs) and is proposed to be the largest intravascular storage pool of vasoactive NO. The mechanism by which nitrite exerts NO vasoactivity remains unclear but deoxyHb exhibits nitrite reductase activity. NitrosylHb (HbFe(II)NO) is formed on nitrite reduction by excess deoxyHb, and S-nitrosated Hb (HbSNO) has also been detected in nitrite/deoxyHb incubations. We report data consistent with efficient HbSNO generation from a nitrosylHb intermediate on oxygenation of anaerobic deoxyHb incubations containing physiologically revelant levels of nitrite, whereas previously a labile nitrosylmetHb (HbFe(III)NO) transient was proposed. The HbSNO yield as a function of the initial nitrite concentration varies with the nitrite/deoxyHb ratio, the incubation time, the concentration of added metHb (a nitrite trap), and the concentration of added cyanide (a strong metHb ligand). Our results reveal that metHb strongly attenuates HbSNO formation, which suggests that the met protein may play a regulatory role by limiting the amount of free (or non-Hb-bound) nitrite within RBCs to prevent hypotension.     

Interactions of haemoglobin with the Neisseria meningitidis receptor HpuAB: the role of TonB and an intact proton motive force

We have characterized the interaction of the Neisseria meningitidis TonB-dependent receptor HpuAB with haemoglobin (Hb). Protease accessibility assays indicated that HpuA and HpuB are surface exposed, HpuB interacts physically with HpuA, and TonB energization affects the conformation of HpuAB. Binding assays using [125I]-Hb revealed that the bipartite receptor has a single binding site for Hb (Kd 150 nM). Competitive binding assays using heterologous Hbs revealed that HpuAB Hb recognition was not species specific. The binding kinetics of Hb to HpuAB were dramatically altered in a TonB- mutant and in wild-type meningococci treated with the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that TonB and an intact proton motive force are required for normal Hb binding and release from HpuAB. Our results support a model in which both HpuA and HpuB are required to form a receptor complex in the outer membrane with a single binding site, whose structure and ligand interactions are significantly affected by the TonB-mediated energy state of the receptor.     

Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis

Hemoglobin (Hb) potently inactivates the nitric oxide (NO) radical via a dioxygenation reaction forming nitrate (NO(3)(-)). This inactivation produces endothelial dysfunction during hemolytic conditions and may contribute to the vascular complications of Hb-based blood substitutes. Hb also functions as a nitrite (NO(2)(-)) reductase, converting nitrite into NO as it deoxygenates. We hypothesized that during intravascular hemolysis, nitrite infusions would limit the vasoconstrictive properties of plasma Hb. In a canine model of low- and high-intensity hypotonic intravascular hemolysis, we characterized hemodynamic responses to nitrite infusions. Hemolysis increased systemic and pulmonary arterial pressures and systemic vascular resistance. Hemolysis also inhibited NO-dependent pulmonary and systemic vasodilation by the NO donor sodium nitroprusside. Compared with nitroprusside, nitrite demonstrated unique effects by not only inhibiting hemolysis-associated vasoconstriction but also by potentiating vasodilation at plasma Hb concentrations of <25 muM. We also observed an interaction between plasma Hb levels and nitrite to augment nitroprusside-induced vasodilation of the pulmonary and systemic circulation. This nitrite reductase activity of Hb in vivo was recapitulated in vitro using a mitochondrial NO sensor system. Nitrite infusions may promote NO generation from Hb while maintaining oxygen delivery; this effect could be harnessed to treat hemolytic conditions and to detoxify Hb-based blood substitutes.     

Structural requirements for hemoglobin to induce fibronectin receptor expression in Candida albicans

Hemoglobin (Hb) is a host factor that induces expression of a promiscuous receptor on Candida albicans for fibronectin (FN) and several other extracellular matrix proteins. FN receptor expression was induced by ferric (Hb(+)Met and Hb(+)CN), ferrous (HbCO and HbO(2)), and cobalt-protoporphyrin derivatives of Hb, whereas globin was inactive. The Hb derivatives all exhibited saturable, dose-dependent kinetics of FN receptor induction, suggesting that Hb may be acting as a receptor ligand. Soluble Hb bound saturably to a low-affinity binding site [K(d) = (1.1+/-0.2) x 10(-6) M] on C. albicans blastospores. However, uptake of (55)FeHb revealed that heme or iron transport into the cell is not required for induction, since internalization of (55)Fe from Hb did not occur until after induction of FN binding. The serum Hb-binding protein, haptoglobin, specifically abrogated this response, indicating that protein structure rather than the heme ligand or iron is necessary for induction of this signaling pathway. C. albicans also adhered to immobilized Hb, which was sufficient to induce FN receptor expression, and to Hb polymers that formed in defined Hb liquid media in the presence of cells. Formation of Hb polymers in solution required metabolic energy, since the aggregation process was halted with azide addition. Collectively, these data demonstrate that C. albicans recognizes polymerized Hb through multivalent low-affinity interactions, and this may be a host environmental cue that triggers extracellular matrix receptor expression at a septic site.     

Haptoglobin Preferentially Binds β but Not α Subunits Cross-Linked Hemoglobin Tetramers with Minimal Effects on Ligand and Redox Reactions

Human hemoglobin (Hb) and haptoglobin (Hp) exhibit an extremely high affinity for each other, and the dissociation of Hb tetramers into dimers is generally believed to be a prerequisite for complex formation. We have investigated Hp interactions with native Hb, αα, and ββ cross-linked Hb (ααXLHb and ββXLHb, respectively), and rapid kinetics of Hb ligand binding as well as the redox reactivity in the presence of and absence of Hp. The quaternary conformation of ββ subunit cross-linking results in a higher binding affinity than that of αα subunit cross-linked Hb. However, ββ cross-linked Hb exhibits a four fold slower association rate constant than the reaction rate of unmodified Hb with Hp. The Hp contact regions in the Hb dimer interfaces appear to be more readily exposed in ββXLHb than ααXLHb. In addition, apart from the functional changes caused by chemical modifications, Hp binding does not induce appreciable effects on the ligand binding and redox reactions of ββXLHb. Our findings may therefore be relevant to the design of safer Hb-based oxygen therapeutics by utilizing this preferential binding of ββXLHb to Hp. This may ultimately provide a safe oxidative inactivation and clearance pathway for chemically modified Hbs in circulation.     

Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine

Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)     

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α(2) β(2) (Pro100Leu) )

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O(2) affinity compared with normal cells (P(50) = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O(2) affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO(2) , decreasing the rate approximately twofold. These kinetic data help explain the high O(2) affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure.     

A study on the quaternary structure change of hemoglobin in the ligation process

In order to inquire into the molecular mechanism underlying the cooperative ligand binding to hemoglobin (Hb), conformational interaction at the interfaces between subunits are investigated on the basis of the atomic coordinates of human deoxy and human carbonmonoxy Hbs. Hypothetical intermediate structures are used, each of which is obtained from the procedure where one or more subunits in deoxy Hb are replaced by the corresponding CO-liganded subunits in carbonmonoxy Hb using the method of superimposition of two sets of atomic coordinates. When either alpha or beta subunit is substituted with the corresponding subunit in carbonmonoxy Hb, serious steric hindrances are produced between alpha 1FG4(92)Arg and beta 2C3(37)Trp or between alpha 1C6(41)Thr and beta 2FG4(97)His, all of which belong to the allosteric core affected directly by ligand binding. These steric hindrances become more serious when both alpha 1(alpha 2) and beta 2(beta 1) subunits are substituted. Therefore the change in the relative distance between iron atom and porphyrin by ligation results in strain in the C-terminal residues as an effect of the steric hindrance between the FG and C segments. However, no steric hindrance can be seen between subunits when the subunits in carbonmonoxy Hb are substituted with the corresponding subunits in deoxy Hb. The nature of the quaternary structural change from liganded to deoxy Hb seems to be different from that from deoxy to liganded Hb.     

Ethylisocyanide equilibria of hemoglobins M Iwate, M Boston, M Hyde Park, M Saskatoon, and M Milwaukee-I in half-ferric and fully reduced states.

The ethylisocyanide equilibria of all the five known hemoglobins M, namely Hb M Iwate (alpha287 Tyrbeta2), Hb M Boston (alpha258 Tyrbeta2), Hb M Hyde Park (alpha2beta292 Tyr), Hb M Saskatoon (alpha2beta263 tyr), and Hb M Milwaukee-I (alpha2beta267 Glu), were studied both in the half-ferric and fully reduced heme states. In the half-ferric state, no heme-heme interaction was observed for Hb M Iwate, Hb M Boston, and Hb M Hyde Park, but Hb M Saskatoon and Hb M Milwaukee-I show small but definite heme-heme interaction with Hill's n of 1.3. The beta chain mutants, Hb M Hyde Park and Hb M Saskatoon, have almost normal affinity for ethylisocyanide and a normal Bohr effect, whereas the alpha chain mutants, Hb M Iwate and Hb M Boston, have abnormally low affinity and almost no Bohr effect. Hb M Milwaukee-I showed a large Bohr effect and low affinity. These results are consistent qualitatively with those on oxygen equilibria reported previously. In the fully reduced state, in which all four hemes were in the ferrous state and capable of binding ethylisocyanide distinct differences were found in the extent of heme-heme interaction. Namely, the n values for proximal histidine mutants, Hb M Iwate and Hb M Hyde Park, were 1.1 and 1.0, respectively, whereas the distal histidine mutants, Hb M Boston and Hb M Saskatoon, showed high n values of 2.4 and 1.6, respectively. Hb M Milwaukee-I also exhibited a high n value of 2.0 The ethylisocyanide affinity of the four histidine mutants was high compared with that of Hb A, while that for Hb M Milwaukee-I was almost normal. All five Hbs M had approximately normal magnitudes of Bohr effect. In the half-ferric state, the proximal and distal histidine mutants of the same chain showed similar affinity for ethylisocyanide and Bohr effect, rather different from those of the mutants of the opposite chain. These differences seem to be derived from the difference of abnormal bonding of ferric iron to tyrosine or glutamic acid. On the other hand, the reduction of iron, which abolished the abnormal bonding and made all of the chains capable of binding ligand, extinguished the differences of alpha and beta chains, and the effect of amino acid side chains close to iron on ligand binding properties became clear. Proximal histidine, which is considered to trigger the transition between the T and R states, seems to be essential to the heme-heme interaction.     

Alternate substrate binding modes to two mutant (D98N and H255N) forms of nitrite reductase from Alcaligenes faecalis S-6: structural model of a transient catalytic intermediate

High-resolution nitrite soaked oxidized and reduced crystal structures of two active site mutants, D98N and H255N, of nitrite reductase (NIR) from Alcaligenes faecalis S-6 were determined to better than 2.0 A resolution. In the oxidized D98N nitrite-soaked structures, nitrite is coordinated to the type II copper via its oxygen atoms in an asymmetric bidentate manner; however, elevated B-factors and weak electron density indicate that both nitrite and Asn98 are less ordered than in the native enzyme. This disorder likely results from the inability of the N delta 2 atom of Asn98 to form a hydrogen bond with the bound protonated nitrite, indicating that the hydrogen bond between Asp98 and nitrite in the native NIR structure is essential in anchoring nitrite in the active site for catalysis. In the oxidized nitrite soaked H255N crystal structure, nitrite does not displace the ligand water and is instead coordinated in an alternative mode via a single oxygen to the type II copper. His255 is clearly essential in defining the nitrite binding site despite the lack of direct interaction with the substrate in the native enzyme. The resulting pentacoordinate copper site in the H255N structure also serves as a model for a proposed transient intermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinated to the copper. The formation of an unusual dinuclear type I copper site in the reduced nitrite soaked D98N and H255N crystal structures may represent an evolutionary link between the mononuclear type I copper centers and dinuclear Cu(A) sites.     

Coarse‐grained and all‐atom modeling of structural states and transitions in hemoglobin

Hemoglobin (Hb), an oxygen‐binding protein composed of four subunits (α1, α2, β1, and β2), is a well‐known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse‐grained (CG) modeling, all‐atom simulation, and structural data from X‐ray crystallography and wide‐angle X‐ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot‐spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all‐atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all‐atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite

The clam Lucina pectinalis supports its symbiotic bacteria by H₂S transport in the open and accessible heme pocket of Lucina Hb I and by O₂ transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O₂ was removed from its active site. Structural analysis revealed that O₂ “clams up” the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O₂ affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O₂. These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins.