Bohr Effect Research Articles

Life on the edge: O2 binding in Atlantic cod red blood cells near their southern distribution limit is not sensitive to temperature or haemoglobin genotype.

ABSTRACTAtlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O2 saturation difference, Sa–vO2, another major determinant of circulatory O2 supply rate. The results showed statistically indistinguishable red blood cell O2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O2 binding (Bohr and Root effects). Modelling of Sa–vO2 at physiological pH, temperature and O2 partial pressures revealed a substantial capacity for increases in Sa–vO2 to meet rising tissue O2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa–vO2 with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O2 supply to tissue demand.

Biological Semiconductors: Structural Control of Heme Redox Potentials in PpcA, a 3-Heme Cytochrome

In an effort to tune and optimize relative bandgap potentials found in biological nanowires that form from multiheme domains of members of the cytochrome c7 family, we developed and extensively characterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 family native to Geobacter sulfurreducens. These mutations were engineered to influence the redox potential (Em) of the middle heme (heme III) in PpcA by using four different strategies: performing charge reversal mutations, decreasing solvent access to the heme plane with bulky residues, altering the native bis-histidine axial ligation of the heme, and by attempting to form hydrogen bonds with the propionates of the heme. The latter strategy is expected not only to increase Em but also to introduce a redox Bohr effect. Out of 12 mutants, 11 were successfully expressed in E.coli and show thermal stability in temperature-dependent CD experiments comparable to the wild-type protein (Tm > 90°C). HPLC-ESI-MS was used to confirm both the purity and the mass of the expressed mutants. Small-angle X-ray scattering confirmed that the mutant proteins formed the expected compact globular structures. In addition to ongoing attempts to obtain protein crystals for X-ray crystallography, we performed extensive all-atom molecular dynamics simulations for all mutants. Formation of propionate hydrogen bonds is supported by simulations for A19R and A23R, while increased solvent access is supported for A19I, A19R, A23R, H20M, and K60E. UV/Vis and NMR redox titrations were performed in order to measure the effect of the mutations on the electrochemical properties of all 3 hemes and to understand the underlying principles and viable approaches in tuning relative heme redox potentials. Successful development of this project may lead to biological semiconductors with much smaller footprints and selectively tunable bandgap properties.

AltitudeOmics: Red Blood Cell Metabolic Adaptation to High Altitude Hypoxia.

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia through the so-called oxygen-dependent metabolic regulation, which involves the competitive binding of deoxyhemoglobin and glycolytic enzymes to the N-terminal cytosolic domain of band 3. This mechanism promotes the accumulation of 2,3-DPG, stabilizing the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the Bohr effect. Despite in vitro studies, in vivo adaptations to hypoxia have not yet been completely elucidated. Within the framework of the AltitudeOmics study, erythrocytes were collected from 21 healthy volunteers at sea level, after exposure to high altitude (5260 m) for 1, 7, and 16 days, and following reascent after 7 days at 1525 m. UHPLC-MS metabolomics results were correlated to physiological and athletic performance parameters. Immediate metabolic adaptations were noted as early as a few hours from ascending to >5000 m, and maintained for 16 days at high altitude. Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide, and sulfur/H2S metabolism. Metabolic adaptations were preserved 1 week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Unveiling the Structural Basis That Regulates the Energy Transduction Properties within a Family of Triheme Cytochromes from Geobacter sulfurreducens.

A family of triheme cytochromes from Geobacter sulfurreducens plays an important role in extracellular electron transfer. In addition to their role in electron transfer pathways, two members of this family (PpcA and PpcD) were also found to be able to couple e-/H+ transfer through the redox Bohr effect observed in the physiological pH range, a feature not observed for cytochromes PpcB and PpcE. In attempting to understand the molecular control of the redox Bohr effect in this family of cytochromes, which is highly homologous both in amino acid sequence and structures, it was observed that residue 6 is a conserved leucine in PpcA and PpcD, whereas in the other two characterized members (PpcB and PpcE) the equivalent residue is a phenylalanine. To determine the role of this residue located close to the redox Bohr center, we replaced Leu6 in PpcA with Phe and determined the redox properties of the mutant, as well as its solution structure in the fully reduced state. In contrast with the native form, the mutant PpcAL6F is not able to couple the e-/H+ pathway. We carried out the reverse mutation in PpcB and PpcE (i.e., replacing Phe6 in these two proteins by leucine) and the mutated proteins showed an increased redox Bohr effect. The results clearly establish the role of residue 6 in the control of the redox Bohr effect in this family of cytochromes, a feature that could enable the rational design of G. sulfurreducens strains that carry mutant cytochromes with an optimal redox Bohr effect that would be suitable for various biotechnological applications.

Bohr effect of avian hemoglobins: Quantitative analyses based on the Wyman equation

The Bohr effect data for bar-headed goose, greylag goose and pheasant hemoglobins can be fitted with the Wyman equation for the Bohr effect, but under one proviso: that the pKa of His146β does not change following the T→R quaternary transition. This assumption is based on the x-ray structure of bar-headed goose hemoglobin, which shows that the salt-bridge formed between His146β and Asp94β in human deoxyhemoglobin is not formed in goose deoxyhemoglobin. When the Bohr data for chicken hemoglobin were fitted by making the same assumption, the pKa of the NH3+ terminal group of Val1α decreased from 7.76 to 6.48 following the T→R transition. When the data were fitted without making any assumption, the pKa of the NH3+ terminal group increased from 7.57 to 7.77 following the T→R transition. We demonstrate that avian hemoglobin Bohr data are readily fitted with the Wyman equation because avian hemoglobins lack His77β. From curve-fitting to Bohr data we estimate the pKas of the NH3+ terminal group of Val1α in the R and T states to be 6.33±0.1 and 7.22±0.1, respectively. We provide evidence indicating that these pKas are more accurate than estimates from kinetic studies.

Two methods to adapt the human haemoglobin–oxygen dissociation algorithm to the blood of white rhinoceros (Ceratotherium simum) and to determine the accuracy of pulse oximetry

ObjectivesTo adapt the algorithm for the calculation of oxygen saturation to the blood characteristics of the white rhinoceros by two different methods and to determine the accuracy of conventional pulse oximetry measurements. Study designAdaptation of two mathematical models of the oxygen dissociation curve (ODC). AnimalsTwenty-five captive white rhinoceros (Ceratotherium simum), including 12 males and 13 females, aged 6–32 years. MethodsDuring 33 anaesthetic events, 94 arterial blood gas samples with 72 simultaneous pulse oximetry measurements were analysed. The calculation of oxygen saturation was adapted to the characteristics of rhinoceros blood using two different methods. Firstly, a mathematical model developed in 1984 and, secondly, an oxygen status algorithm (OSA) produced by the same developer in 2005 were tested for their applicability for clinical use. ResultsWhen arterial partial pressure of oxygen is >7.98 kPa (60 mmHg), oxygen saturation exceeds 95%. At partial pressures of 6.12–6.52 kPa (46–49 mmHg) Method 1 determined oxygen saturations of 92.5–95.3% and Method 2 oxygen saturations of 90.2–91.6%. Both methods resulted in similar ODCs and accounted for the low p50 value of rhinoceros blood. Method 1 provided better adaptation in respect to the physiological parameters of the rhinoceros, especially with regard to the Bohr effect, than Method 2. Pulse oximetry was an unreliable method of monitoring arterial oxygen saturation during general anaesthesia in this species. ConclusionAdapting the oxygen saturation algorithm to consider the left shift of the ODC provides a useful tool for monitoring oxygen status, especially as pulse oximetry is insufficiently accurate. Experimental determination of the complete Hill curve is required to further validate and optimize the algorithm for use in the white rhinoceros. Clinical relevanceThe method will facilitate the accurate interpretation of oxygen saturation calculated by blood gas analysis in white rhinoceros.

In vitro effects of increased temperature and decreased pH on blood oxygen affinity of 10 fish species of the Amazon.

Blood-O2 affinities (P50 ) were measured over a physiologically relevant pH range at 31 (highest temperature average of Rio Negro over the last 8 years), 33 and 35° C for 10 species of the Rio Negro, aiming to test the acute effects of temperature foreseen by the IPCC (Intergovernmental Panel on Climate Change) for coming years. The animals were collected during an expedition to the Anavilhanas Islands of the Rio Negro, 110 km upstream from Manaus (2° 23' 41″ S; 60° 55' 14″ W). Hoplias malabaricus showed higher blood-O2 sensitivity to pH changes (Bohr effect, Φ = Δlog10 P50 ΔpH(-1) ) at both 31° C (Φ = -0·44) and 35° C (Φ = -0·26) compared to Osteoglossum bicirrhosum (Φ = -0·54 at 31° C and Φ = -0·58 at 35° C), but lower P50 under most conditions, and a greater sensitivity of P50 to temperature. Two out of the 10 analysed species had significant increases of P50 (lower blood-O2 affinity) at the highest temperature throughout the pH range tested. For all other species, a minor increase of P50 over the assay-tested temperatures was observed, although all presented a normal Bohr effect. Overall, a diversity of intensities of pH and temperature effects on blood-O2 affinities was observed, which seems to be connected to the biological characteristics of the analysed species. Thermal disturbances in their habitats, likely to occur due to the global warming, would impair blood-O2 binding and unloading in some of the analysed fish species. Copyright © 2016 John Wiley & Sons, Ltd.

Carbon Dioxide–Assisted Pneumodissection in Fat Grafting

Fat grafting has become a popular procedure due to its wide range of clinical applications. Several fat grafting techniques have been described to increase the survival rate of the grafted fat. This article presents carbon dioxide (CO2) pneumodissection to improve the biological microenvironment of the recipient site. A total of 40 patients were treated with CO2 pneumodissection for aesthetic and reconstructive indications. The known effects of carboxitherapy such as vasodilation, enhanced Bohr effect, and neoangiogenesis are applied to facilitate the grafting procedure and in the search for higher rate of graft uptake. Tissue pneumodissection has demonstrated reduced pain due to the fat injection process, and reduces the sedation requirements and local anesthesia infiltration requirements.

Oxygen binding affinities and effector sensitivities of hemoglobins of African mole rats inhabiting hypoxic and hypercapnic burrows

African mole rats (family Bathyergidae) that excavate and inhabit deep, complex and sealed underground galleries may become subjected to severe hypoxia and hypercapnia (O2 tensions falling below that at the summit of Mount Everest, and CO2 tensions up to 10% of barometric pressure) that predictably impose austere constraints on the exchange and internal transport of respiratory gases. In contrast to high‐altitude mammals, little information is available on possible adaptations in hemoglobin (Hb) function that may mitigate these constraints. Aiming to identify the cellular and molecular mechanisms that safeguard tissue O2 supply in these mole rats, we measured the hematological characteristics as well as Hb‐O2 affinity and it's sensitivity to pH (the Bohr effect), CO2 tension, 2,3‐diphosphoglycerate (DPG, the major organic cofactor modulating Hb‐O2 affinity in mammalian red cells) and to temperature, in six (two strictly‐solitary and four social) species of mole rats originating from a range of Southern African biomes and soil types. The Hbs of the different species showed slight variation in O2 affinity and its sensitivity to pH, DPG and temperature, that did not correlate with soil‐type and sociality, but exhibited conspicuous and variable reductions in specific (pH‐independent) CO2 effects compared to other mammalian Hbs, which may be of adaptive significance in safeguarding pulmonary Hb‐O2 loading in hypercapnic burrows. The observations align with the contention that blood O2‐affinities of burrowing mammals are pre‐adapted to high altitude. Attempts are made to correlate the measured oxygenation traits with available amino acid sequences for mole rat Hbs.

Seasonal variations of functional properties of hemoglobin and ionic environment in the Freshwater fish: An example of bream, Abramis brama (Cyprinidae)

The dynamics of hemoglobin affinity to oxygen, Bohr effect value, and concentrations of sodium, potassium, calcium and magnesium were studied in the bream, Abramis brama, erythrocytes of the Rybinsk reservoir during an annual cycle. The studied parameters were most variable during the fish reproductive period. On the basis of the original studies, as well as of some published data concerning physiological–biochemical parameters, it is suggested that the state of spawning is comparable to the state of stress. The relation between the seasonal dynamics of hemoglobin functional properties and the parameters of ionic environment is revealed.

Bohr effect and temperature sensitivity of hemoglobins from highland and lowland deer mice

An important means of physiological adaptation to environmental hypoxia is an increased oxygen (O2) affinity of the hemoglobin (Hb) that can help secure high O2 saturation of arterial blood. However, the trade-off associated with a high Hb–O2 affinity is that it can compromise O2 unloading in the systemic capillaries. High-altitude deer mice (Peromyscus maniculatus) have evolved an increased Hb–O2 affinity relative to lowland conspecifics, but it is not known whether they have also evolved compensatory mechanisms to facilitate O2 unloading to respiring tissues. Here we investigate the effects of pH (Bohr effect) and temperature on the O2-affinity of high- and low-altitude deer mouse Hb variants, as these properties can potentially facilitate O2 unloading to metabolizing tissues.Our experiments revealed that Bohr factors for the high- and low-altitude Hb variants are very similar in spite of the differences in O2-affinity. The Bohr factors of deer mouse Hbs are also comparable to those of other mammalian Hbs. In contrast, the high- and low-altitude variants of deer mouse Hb exhibited similarly low temperature sensitivities that were independent of red blood cell anionic cofactors, suggesting an appreciable endothermic allosteric transition upon oxygenation. In conclusion, high-altitude deer mice have evolved an adaptive increase in Hb–O2 affinity, but this is not associated with compensatory changes in sensitivity to changes in pH or temperature. Instead, it appears that the elevated Hb–O2 affinity in high-altitude deer mice is compensated by an associated increase in the tissue diffusion capacity of O2 (via increased muscle capillarization), which promotes O2 unloading.

The effects of hypercapnia on the West Coast rock lobster (Jasus lalandii) through acute exposure to decreased seawater pH — Physiological and biochemical responses

The cold water palinurid Jasus lalandii (“West Coast rock lobster”) is a commercially important crustacean in South Africa and Namibia and inhabits the Benguela Current Eastern Boundary System. This habitat is characterised by strong upwelling events in summer and algal blooms with their subsequent decay in autumn. Upwelling can lead to acute hypercapnia whereas the algal decay is associated with acute hypercapnic hypoxia. Both types of hypercapnic events could become more frequent and severe in the future due to ongoing climate change. The aim of the present study was, however, to study the capability and mechanisms of response in J. lalandii to hypercapnia exclusively. Accordingly, the following research questions were formulated: 1) To what extent is haemocyanin oxygen-binding affinity of adult J. lalandii pH-sensitive? 2) Can adult male J. lalandii respond swiftly to drastic changes in pH? 3) What physiological mechanisms facilitate a potential response to a drastically declining pH, i.e. acute hypercapnia? These questions were answered by analysing 1) the pH sensitivity of the haemocyanin's oxygen binding properties and 2) in vivo changes in the acid–base balance of adult J. lalandii during acute exposure to hypercapnia (pH7.4). Results showed the following: 1) Haemocyanin displays a strong Bohr shift (whole haemolymph: ΔlogP50/ΔpH=−1.17; dialysed haemolymph: ΔlogP50/ΔpH=−0.84) in response to lowering of pH. 2) Acute hypercapnia leads to a decline in extracellular pH within the initial 1.5h of exposure. 3) Thereafter, active compensation becomes apparent as the bicarbonate levels start to increase, with complete compensation reached after 5h of exposure (+2.3mmoll−1; +48%). 3) This bicarbonate increase is reversed when returning lobsters to normocapnia (pH7.9). 4) Levels of molecular modulators of haemocyanin oxygen affinity (Ca2+, Mg2+ and l-lactate) do not change during acute exposure to hypercapnia.Our results show the capability of adult J. lalandii to rapidly and fully compensate the experienced extracellular acidosis, protecting oxygen carrying capacity of haemocyanin and ensuring an outward gradient of CO2. The West Coast rock lobster is therefore well equipped for its habitat where these hypercapnic events are known to occur frequently.

CD73 and AMPD3 deficiency enhance metabolic performance via erythrocyte ATP that decreases hemoglobin oxygen affinity.

Erythrocytes are the key target in 5′-AMP induced hypometabolism. To understand how regulation of endogenous erythrocyte AMP levels modulates systemic metabolism, we generated mice deficient in both CD73 and AMPD3, the key catabolic enzymes for extracellular and intra-erythrocyte AMP, respectively. Under physiological conditions, these mice displayed enhanced capacity for physical activity accompanied by significantly higher food and oxygen consumption, compared to wild type mice. Erythrocytes from Ampd3−/− mice exhibited higher half-saturation pressure of oxygen (p50) and about 3-fold higher levels of ATP and ADP, while they maintained normal 2,3-bisphosphoglycerate (2,3-BPG), methemoglobin levels and intracellular pH. The affinity of mammalian hemoglobin for oxygen is thought to be regulated primarily by 2,3-BPG levels and pH (the Bohr effect). However, our results show that increased endogenous levels of ATP and ADP, but not AMP, directly increase the p50 value of hemoglobin. Additionally, the rise in erythrocyte p50 directly correlates with an enhanced capability of systemic metabolism.

Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (βE6V/αH20R) and rHb (βE6V/αH20Q)) or lateral (rHb (βE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The (1)H NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis-20 or αHis-50 do not change the subunit interfaces or the heme pockets of the proteins. The double mutants show only slight structural alteration in the β-heme pockets. All mutants have similar cooperativity (n50), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P50) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (βE6V/αH20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 ± 0.20 g/dl. Hb S, rHb (βE6V/αH20Q), rHb (βE6V/αH50Q), rHb (βE6V/αH20R/αH50Q), and rHb (βE6V/αH20Q/αH50Q) have dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66, and 10.89 ± 0.83 g/dl, respectively. rHb (βE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C).

Bohr effect of hemoglobins: Accounting for differences in magnitude

The basis of the difference in the Bohr effect of various hemoglobins has remained enigmatic for decades. Fourteen amino acid residues, identical in pairs and located at specific ‘Bohr group positions’ in human hemoglobin, are implicated in the Bohr effect. All 14 are present in mouse, 11 in dog, eight in pigeon and 13 in guinea pig hemoglobin. The Bohr data for human and mouse hemoglobin are identical: the 14 Bohr groups appear at identical positions in both molecules. The dog data are different from the human because three Bohr group positions are occupied by non-ionizable groups in dog hemoglobin; the pigeon data are vastly different from the human because six Bohr group positions are occupied by non-ionizable groups in pigeon hemoglobin. The guinea pig data are quite complex. Quantitative analyses showed that only the pigeon data could be fitted with the Wyman equation for the Bohr effect. We demonstrate that, apart from guinea pig hemoglobin, the difference between the Bohr effect of each of the other hemoglobins and of pigeon hemoglobin can be accounted for quantitatively on the basis of the occupation of some of their Bohr group positions by non-ionizable groups in pigeon hemoglobin. We attribute the anomalous guinea pig result to a new salt-bridge formed in its R2 quaternary structure between the terminal NH3+ group of one β-chain and the COO− terminal group of the partner β-chain in the same molecule. The pKas of this NH3+ group are 6.33 in the R2 and 4.59 in the T state.

Acid–base balance and changes in haemolymph properties of the South African rock lobsters, Jasus lalandii, a palinurid decapod, during chronic hypercapnia

Few studies exist reporting on long-term exposure of crustaceans to hypercapnia. We exposed juvenile South African rock lobsters, Jasus lalandii, to hypercapnic conditions of pH 7.3 for 28 weeks and subsequently analysed changes in the extracellular fluid (haemolymph). Results revealed, for the first time, adjustments in the haemolymph of a palinurid crustacean during chronic hypercapnic exposure: 1) acid-base balance was adjusted and sustained by increased bicarbonate and 2) quantity and oxygen binding properties of haemocyanin changed. Compared with lobsters kept under normocapnic conditions (pH 8.0), during prolonged hypercapnia, juvenile lobsters increased bicarbonate buffering of haemolymph. This is necessary to provide optimum pH conditions for oxygen binding of haemocyanin and functioning of respiration in the presence of a strong Bohr Effect. Furthermore, modification of the intrinsic structure of the haemocyanin molecule, and not the presence of molecular modulators, seems to improve oxygen affinity under conditions of elevated pCO2.

Functional properties of hemoglobin during the adaptation of fish to low environmental pH

The dependence of the resistance of fish to acidification on the functional properties of hemoglobin has been studied in 21 species (nine families) of freshwater, marine, and anadromous fish. A direct correlation between the fish tolerance to low pH and the value of the Bohr effect has been revealed for the first time. On the basis of these data, the Bohr effect is proposed as a characteristic of the buffer properties of hemoglobin and acid resistance in fish. The curves of the oxygen equilibrium of hemoglobin are invariant in the fish having the lowest resistance to acidification (sturgeons). The maximum distortion of the invariance in the acidic buffer solution was observed in the most acid-resistant fish: pike and perch. The efficiency of the hemoglobin buffer system declined with decreasing resistance of fish to acidification.

Investigating alpha-globin structural variants: a retrospective review of 135,000 Brazilian individuals.

BackgroundBrazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population.MethodsA total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC) and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC). Mutations were identified by molecular analyses, and the oxygen affinity, heme–heme cooperativity and Bohr effect of the variants were evaluated by functional tests.ResultsFour new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia.ConclusionThe variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations.

New look at hemoglobin allostery.

New look at hemoglobin allostery.

Role of Ionic Strength and the Bohr Effect in Modulating Thermodynamic Profiles Associated with Co Escape in Rice Non-Symbiotic Hemoglobin 1

Rice hemoglobin (rHb1) is a type 1 non-symbiotic hemoglobin (nsHb1), which is found in plants and which is evolutionarily related to leghemoglobins and truncated hemoglobins. It is structurally similar to vertebrate heme proteins, containing Fe-protoporphyrin IX and the classic 3-over-3 globin fold. Unlike other hexacoordinate heme proteins found in plants, the ligand binding properties of nsHb1 proteins make it unlikely to be involved in nitrogen fixation or oxygen transport. We have characterized ligand migration upon CO photorelease from rHb1 under various conditions in order to elucidate functional role(s) of rHb1 in vivo. Using photoacoustic calorimetry, we have determined thermodynamic parameters (ΔH and ΔV) for CO photorelease from wild type rHb1 and a distal histidine to leucine (H74L) mutant taking place within ∼ 5 µs after ligand photo-dissociation. Under stripped conditions, we observe a strong temperature dependence of the observed parameters. Photorelease of CO from the wild type protein below 16°C is associated with ΔH = 28.7 ± 2.2 kcal mol−1 and ΔV = 4.3 ± 0.4 mL mol−1, while above 16°C we observe ΔH = 9.7 ± 3.4 kcal mol−1 and ΔV = 0.4 ± 0.7 mL mol−1. This temperature dependence is not observed in the H74L mutant, in the presence of 500 mM NaCl, or at pH 6. We have also determined rate constants and quantum yields for bimolecular rebinding of CO using transient absorption spectroscopy, both of which increase with temperature. The contributions of electrostriction, proton uptake by Bohr groups, oligomerization state, and the distal residue of rHb1 to the observed kinetic and thermodynamic parameters will be discussed. We will also discuss ligand migration pathways and protein dynamics upon CO photorelease in rHb1.