Domain-Specific Effector Interactions within the Central Cavity of Human Adult Hemoglobin in Solution and in Porous Sol−Gel Matrices:  Evidence for Long-Range Communication Pathways<sup>,</sup>

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Chem. 277, 34508 -34520). To further investigate this effect in more detail, ␣-and ␤-semihemoglobins, namely, ␣(heme)␤(apo) and ␣(apo)␤(heme), respectively, were prepared and characterized with respect to the impact of allosteric effectors on both conformation and ligand binding properties. Semihemoglobins are dimers characterized by a high affinity for oxygen and lack of cooperativity. We found that, compared with stripped conditions, semihemoglobins responded to effectors (inositol hexaphosphate and L35) by decreasing the affinity for oxygen by 60-and 130-fold for ␣-and ␤-semihemoglobins, respectively. 1 H NMR and sedimentation velocity experiments carried out with their ligated and unligated forms in the absence and presence of effectors revealed that semihemoglobins always remain as singleheme-carrying dimers. Recombination kinetics of their photolyzed CO derivatives showed that effectors did indeed interact with their ligated forms. Measurements of the Fe-His stretching mode show that the semihemoglobins undergo a large ligand binding-induced conformational shift and that both ligand-free and ligand derivatives respond to the presence of effectors. Contradictions to the Monod-Wyman-Changeaux/Perutz allosteric model arise since 1) the modulation of ligand affinity is not achieved in semihemoglobins by the formation of a low affinity T conformation (quaternary effect) but by direct interaction with effectors, 2) effectors do interact significantly with ligated forms of high affinity semihemoglobins, and 3) modulation of the ligand affinity and the cooperativity are not necessarily linked but instead can be separated into two distinct phenomena that can be isolated.Hemoglobin (Hb), 1 the oxygen transport protein in blood, is a tetrameric molecule composed by the assembly of two identical ␣␤ heterodimers in which each subunit carries a heme group to which oxygen binds reversibly. This ligation process is characterized by a phenomenon called cooperativity; each successive ligation of the hemes within a tetramer is accompanied by a progressive increase in the affinity for oxygen of the remaining hemes. Cooperativity can be described by the so-called twostate or Monod-Wyman-Changeaux (MWC) allosteric model (1), which proposes that Hb is allowed to assume only two conformations, T or tense, characterized by a low affinity for the ligand and typical of the unligated form, and R or relaxed, with high affinity and typical of the ligated form. Progressive ligation shifts eventually the allosteric equilibrium from T to R.With the proposal by Perutz (2) of his stereochemical mechanism, the correlation of two distinct molecular structures, unligated and ligated, with the so-called quaternary T and quaternary R functional conformations, respectively, was tacitly established. The T conformation was stabilized by an intricate network of interdimeric interactions, namely, salt bridges and hydrogen bonds formed mainly between ␣1 and ␤2 (or ␣2 and ␤1). Upon ligation of the hemes, these interactions broke apart, releasing the al...

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confidence: 59%

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confidence: 96%

Semihemoglobins, High Oxygen Affinity Dimeric Forms of Human Hemoglobin Respond Efficiently to Allosteric Effectors without Forming Tetramers

Tsuneshige

Kanaori

Samuni

et al. 2004

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“…There are three potential roles for effectors within this picture. For the deoxy T-state, effectors do not change the equilibrium distribution of conformations (21,61). They do, however, decrease the amplitude of thermally driven equilibrium fluctuations that allow for interconversion among the members of the equilibrium distribution of conformations.…”

Section: Discussionmentioning

confidence: 92%

“…The very initial rate at which nitrite reacts with a heme in a fully deoxy T-state species under a given set of solution conditions is dominated by the high proximal strain and steric hindrance limiting both reactivity with and access to the iron. Modulation of this pattern through effectors occurs through damping of motions that transiently decrease proximal strain and increase accessibility but not by changing the actual equilibrium distribution of LT species within the deoxy-HbA population (21,61). Once a given heme site undergoes ligand binding (as either aquo-met, met-NO, or ferrous-NO), the overall tertiary conformation begins to evolve in a manner that enhances reactivity at the remaining sites (probably within the same ␣␤ dimer (64 -66)).…”

Section: Discussionmentioning

confidence: 99%

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Roche

Dantsker

Samuni

et al. 2006

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Nitrite reductase activity of deoxyhemoglobin (HbA) in the red blood cell has been proposed as a non-nitric-oxide synthase source of deliverable nitric oxide (NO) within the vasculature. An essential element in this scheme is the dependence of this reaction on the quaternary/tertiary structure of HbA. In the present work sol-gel encapsulation is used to trap and stabilize deoxy-HbA in either the T or R quaternary state, thus allowing for the clear-cut monitoring of nitrite reductase activity as a function of quaternary state with and without effectors. The results indicate that reaction is not only R-T-dependent but also heterotropic effector-dependent within a given quaternary state. The use of the maximum entropy method to analyze carbon monoxide (CO) recombination kinetics from fully and partially liganded sol-gel-encapsulated T-state species provides a framework for understanding effector modulation of T-state reactivity by influencing the distribution of high and low reactivity T-state conformations.The physiological role of the nitrite ion is currently attracting considerable research interest arising primarily from observations that indicate the anion can function as a non-nitric-oxide synthase source of nitric oxide (NO) (1-6). It has also been suggested that hemoglobin (Hb) 2 within red blood cells may be a source of deliverable nitrite-derived NO, generated from Hb nitrite reductase activity (deoxy-Hb ϩ nitrite 3 met-Hb ϩ NO) (6 -10). Such a mechanism also has clear implications for the vasoactivity of acellular hemoglobin-based blood substitutes. Although there are studies that support the physiological role of a hemoglobin-based source of nitrite-derived NO, there are still fundamental mechanistic questions that remain unanswered including the key issue of how NO, once bound to a ferrous heme, can be efficiently delivered (11, 12).Studies show that there is a relationship between the hemoglobin P50 and nitrite reductase activity (2, 3, 13, 14) as well as S-nitrosothiol synthase function (14). This result has led to the hypotheses that red blood cell/Hb enzymatic pathways can modulate blood flow and play a role in regulating hypoxic vasodilation (3, 14). The results also imply a relationship between the conformational properties of Hb and nitrite reductase activity (2, 5, 6, 15) and S-nitrosothiol synthase function (14). Direct measurements of the nitrite reductase activity of deoxy-Hb as a function of added allosteric effectors and mutagenic/chemical modifications support the concept of conformational control of Hb nitrite reductase reactivity with the T-state conformation having reduced reactivity compared with R-state conformations of Hb (5, 6). Two limitations of these studies are the inability to stabilize and compare the T and R-state forms of deoxy-HbA (human adult hemoglobin) without mutagenic or chemical modification and the complexity of the reductase reaction in solution due to autoacceleration arising from the allosteric nature of Hb reactivity.The present study directly addresses the qu...

“…The L35 was added to enhance the contribution of the T state population and thus allow for a clear distinction between the T and R state NR kinetics at pH 7.4. For this experiment, L35 has the advantage over inositol hexaphosphate and diphosphoglycerate as a T state stabilizer in that it remains a potent effector at the higher pH (74). The NR reaction in solution was followed at 430 nm as described above for the pH 7.0 samples.…”

Section: Nitrite Reductase Activity Ph 74mentioning

confidence: 99%

Structural and Functional Studies Indicating Altered Redox Properties of Hemoglobin E

Roche

Malashkevich

Balazs

et al. 2011

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Hemoglobin (Hb) E (␤-Glu26Lys) remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms; for example, EE individuals exhibit a mild chronic anemia, and HbE/␤-thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac dysfunction.The purpose of this study was to determine and evaluate structural and functional consequences of the HbE mutation that might account for the pathophysiology. Functional studies indicate minimal allosteric consequence to both oxygen and carbon monoxide binding properties of the ferrous derivatives of HbE. In contrast, redoxsensitive reactions are clearly impacted as seen in the following: 1) the ϳ2.5 times decrease in the rate at which HbE catalyzes nitrite reduction to nitric oxide (NO) relative to HbA, and 2) the accelerated rate of reduction of aquometHbE by L-cysteine (L-Cys). Sol-gel encapsulation studies imply a shift toward a higher redox potential for both the T and R HbE structures that can explain the origin of the reduced nitrite reductase activity of deoxyHbE and the accelerated rate of reduction of aquometHbE by cysteine. Deoxy-and CO HbE crystal structures (derived from crystals grown at or near physiological pH) show loss of hydrogen bonds in the microenvironment of ␤Lys-26 and no significant tertiary conformational perturbations at the allosteric transition sites in the R and T states. Together, these data suggest a model in which the HbE mutation, as a consequence of a relative change in redox properties, decreases the overall rate of Hb-mediated production of bioactive NO.