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Colin J. Brauner - One of the best experts on this subject based on the ideXlab platform.
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The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish
Journal of Comparative Physiology B, 2020Co-Authors: Angelina M Dichiera, Colin J. Brauner, Olivia J. L. Mcmillan, Alexander M. Clifford, Greg G. Goss, Andrew J EsbaughAbstract:In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO_2) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO_2 excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root Effect [where a reduction in pH reduces hemoglobin’s capacity to bind with oxygen (O_2)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.
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Root Effect Haemoglobins in Fish May Greatly Enhance General Oxygen Delivery Relative to Other Vertebrates.
PloS one, 2015Co-Authors: Jodie L. Rummer, Colin J. BraunerAbstract:The teleost fishes represent over half of all extant vertebrates; they occupy nearly every body of water and in doing so, occupy a diverse array of environmental conditions. We propose that their success is related to a unique oxygen (O2) transport system involving their extremely pH-sensitive haemoglobin (Hb). A reduction in pH reduces both Hb-O2 affinity (Bohr Effect) and carrying capacity (Root Effect). This, combined with a large arterial-venous pH change (ΔpHa-v) relative to other vertebrates, may greatly enhance tissue oxygen delivery in teleosts (e.g., rainbow trout) during stress, beyond that in mammals (e.g., human). We generated oxygen equilibrium curves (OECs) at five different CO2 tensions for rainbow trout and determined that, when Hb-O2 saturation is 50% or greater, the change in oxygen partial pressure (ΔPO2) associated with ΔpHa-v can exceed that of the mammalian Bohr Effect by at least 3-fold, but as much as 21-fold. Using known ΔpHa-v and assuming a constant arterial-venous PO2 difference (Pa-vO2), Root Effect Hbs can enhance O2 release to the tissues by 73.5% in trout; whereas, the Bohr Effect alone is responsible for enhancing O2 release by only 1.3% in humans. Disequilibrium states are likely operational in teleosts in vivo, and therefore the ΔpHa-v, and thus enhancement of O2 delivery, could be even larger. Modeling with known Pa-vO2 in fish during exercise and hypoxia indicates that O2 release from the Hb and therefore potentially tissue O2 delivery may double during exercise and triple during some levels of hypoxia. These characteristics may be central to performance of athletic fish species such as salmonids, but may indicate that general tissue oxygen delivery may have been the incipient function of Root Effect Hbs in fish, a trait strongly associated with the adaptive radiation of teleosts.
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Root Effect Hemoglobin May Have Evolved to Enhance General Tissue Oxygen Delivery
Science (New York N.Y.), 2013Co-Authors: Jodie L. Rummer, David J. Mckenzie, Alessio Innocenti, Claudiu T. Supuran, Colin J. BraunerAbstract:The Root Effect is a pH-dependent reduction in hemoglobin-O2 carrying capacity. Specific to ray-finned fishes, the Root Effect has been ascribed specialized roles in retinal oxygenation and swimbladder inflation. We report that when rainbow trout are exposed to elevated water carbon dioxide (CO2), red muscle partial pressure of oxygen (PO2) increases by 65%--evidence that Root hemoglobins enhance general tissue O2 delivery during acidotic stress. Inhibiting carbonic anhydrase (CA) in the plasma abolished this Effect. We argue that CA activity in muscle capillaries short-circuits red blood cell (RBC) pH regulation. This acidifies RBCs, unloads O2 from hemoglobin, and elevates tissue PO2, which could double O2 delivery with no change in perfusion. This previously undescribed mechanism to enhance O2 delivery during stress may represent the incipient function of Root hemoglobins in fishes.
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Plasma-accessible carbonic anhydrase at the tissue of a teleost fish may greatly enhance oxygen delivery: in vitro evidence in rainbow trout, Oncorhynchus mykiss
Journal of Experimental Biology, 2011Co-Authors: Jodie L. Rummer, Colin J. BraunerAbstract:SUMMARY During a generalized acidosis in rainbow trout, catecholamines are released into the blood, activating red blood cell (RBC) Na + /H + exchange (βNHE), thus protecting RBC intracellular pH (pH i ) and subsequent O 2 binding at the gill. Because of the presence of a Root Effect (a reduction in oxygen carrying capacity of the blood with a reduction in pH), the latter could otherwise be impaired. However, plasma-accessible carbonic anhydrase (CA) at the tissues (and absence at the gills) may result in selective short-circuiting of RBC βNHE pH regulation. This would acidify the RBCs and greatly enhance O 2 delivery by exploitation of the combined Bohr-Root Effect, a mechanism not previously proposed. As proof-of-principle, an in vitro closed system was developed to continuously monitor extracellular pH (pH e ) and O 2 tension ( P O 2 ) of rainbow trout blood. In this closed system, adding CA to acidified, adrenergically stimulated RBCs short-circuited βNHE pH regulation, resulting in an increase in P O 2 by >30 mmHg, depending on the starting Hb-O 2 saturation and degree of initial acidification. Interestingly, in the absence of adrenergic stimulation, addition of CA still elevated P O 2 , albeit to a lesser extent, a response that was absent during general NHE inhibition. If plasma-accessible CA-mediated short-circuiting is operational in vivo , the combined Bohr-Root Effect system unique to teleost fishes could markedly enhance tissue O 2 delivery far in excess of that in vertebrates possessing a Bohr Effect alone and may lead to insights about the early evolution of the Root Effect.
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Use it or lose it? Sablefish, Anoplopoma fimbria, a species representing a fifth teleostean group where the βNHE associated with the red blood cell adrenergic stress response has been secondarily lost
Journal of Experimental Biology, 2010Co-Authors: Jodie L. Rummer, Mani Roshan-moniri, Shannon K. Balfry, Colin J. BraunerAbstract:Like most teleosts, sablefish (Anoplopoma fimbria Pallas 1814) blood exhibits a moderate Root Effect (~35% maximal desaturation), where a reduction in blood pH dramatically reduces O(2) carrying capacity, a mechanism important for oxygenating the eye and filling the swim bladder (SB) in teleosts. Although sablefish lack a SB, we observed a well-defined choroid rete at the eye. The adrenergically mediated cell swelling typically associated with a functional red blood cell (RBC) beta-adrenergic Na(+)/H(+) exchanger (betaNHE), which would normally protect RBC pH, and thus O(2) transport, during a generalized acidosis, was not observed in sablefish blood. Neither isoproterenol (a beta-agonist) nor 8-bromo cAMP could elicit this response. Furthermore, RBC osmotic shrinkage, known to stimulate NHEs in general and betaNHE in other teleosts such as trout and flounder, resulted in no significant regulatory volume increase (RVI), further supporting the absence of a functional RBC betaNHE. The onset of the Root Effect occurs at a much lower RBC pH (6.83-6.92) than in other teleosts, and thus RBC betaNHE may not be required to protect O(2) transport during a generalized acidosis in vivo. Phylogenetically, sablefish may represent a fifth group of teleosts exhibiting a secondary reduction or loss of betaNHE activity. However, sablefish have not lost the choroid rete at the eye (unlike in the other four groups), which may still function with the Root Effect to oxygenate the retina, but the low pH onset of the Root Effect may ensure haemoglobin (Hb)-O(2) binding is not compromised at the respiratory surface during a general acidosis in the absence of RBC betaNHE. The sablefish may represent an anomaly within the framework of Root Effect evolution, in that they possess a moderate Root Effect and a choroid rete at the eye, but lack the RBC betaNHE and the SB system.
Jodie L. Rummer - One of the best experts on this subject based on the ideXlab platform.
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Root Effect Haemoglobins in Fish May Greatly Enhance General Oxygen Delivery Relative to Other Vertebrates.
PloS one, 2015Co-Authors: Jodie L. Rummer, Colin J. BraunerAbstract:The teleost fishes represent over half of all extant vertebrates; they occupy nearly every body of water and in doing so, occupy a diverse array of environmental conditions. We propose that their success is related to a unique oxygen (O2) transport system involving their extremely pH-sensitive haemoglobin (Hb). A reduction in pH reduces both Hb-O2 affinity (Bohr Effect) and carrying capacity (Root Effect). This, combined with a large arterial-venous pH change (ΔpHa-v) relative to other vertebrates, may greatly enhance tissue oxygen delivery in teleosts (e.g., rainbow trout) during stress, beyond that in mammals (e.g., human). We generated oxygen equilibrium curves (OECs) at five different CO2 tensions for rainbow trout and determined that, when Hb-O2 saturation is 50% or greater, the change in oxygen partial pressure (ΔPO2) associated with ΔpHa-v can exceed that of the mammalian Bohr Effect by at least 3-fold, but as much as 21-fold. Using known ΔpHa-v and assuming a constant arterial-venous PO2 difference (Pa-vO2), Root Effect Hbs can enhance O2 release to the tissues by 73.5% in trout; whereas, the Bohr Effect alone is responsible for enhancing O2 release by only 1.3% in humans. Disequilibrium states are likely operational in teleosts in vivo, and therefore the ΔpHa-v, and thus enhancement of O2 delivery, could be even larger. Modeling with known Pa-vO2 in fish during exercise and hypoxia indicates that O2 release from the Hb and therefore potentially tissue O2 delivery may double during exercise and triple during some levels of hypoxia. These characteristics may be central to performance of athletic fish species such as salmonids, but may indicate that general tissue oxygen delivery may have been the incipient function of Root Effect Hbs in fish, a trait strongly associated with the adaptive radiation of teleosts.
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Root Effect Hemoglobin May Have Evolved to Enhance General Tissue Oxygen Delivery
Science (New York N.Y.), 2013Co-Authors: Jodie L. Rummer, David J. Mckenzie, Alessio Innocenti, Claudiu T. Supuran, Colin J. BraunerAbstract:The Root Effect is a pH-dependent reduction in hemoglobin-O2 carrying capacity. Specific to ray-finned fishes, the Root Effect has been ascribed specialized roles in retinal oxygenation and swimbladder inflation. We report that when rainbow trout are exposed to elevated water carbon dioxide (CO2), red muscle partial pressure of oxygen (PO2) increases by 65%--evidence that Root hemoglobins enhance general tissue O2 delivery during acidotic stress. Inhibiting carbonic anhydrase (CA) in the plasma abolished this Effect. We argue that CA activity in muscle capillaries short-circuits red blood cell (RBC) pH regulation. This acidifies RBCs, unloads O2 from hemoglobin, and elevates tissue PO2, which could double O2 delivery with no change in perfusion. This previously undescribed mechanism to enhance O2 delivery during stress may represent the incipient function of Root hemoglobins in fishes.
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Plasma-accessible carbonic anhydrase at the tissue of a teleost fish may greatly enhance oxygen delivery: in vitro evidence in rainbow trout, Oncorhynchus mykiss
Journal of Experimental Biology, 2011Co-Authors: Jodie L. Rummer, Colin J. BraunerAbstract:SUMMARY During a generalized acidosis in rainbow trout, catecholamines are released into the blood, activating red blood cell (RBC) Na + /H + exchange (βNHE), thus protecting RBC intracellular pH (pH i ) and subsequent O 2 binding at the gill. Because of the presence of a Root Effect (a reduction in oxygen carrying capacity of the blood with a reduction in pH), the latter could otherwise be impaired. However, plasma-accessible carbonic anhydrase (CA) at the tissues (and absence at the gills) may result in selective short-circuiting of RBC βNHE pH regulation. This would acidify the RBCs and greatly enhance O 2 delivery by exploitation of the combined Bohr-Root Effect, a mechanism not previously proposed. As proof-of-principle, an in vitro closed system was developed to continuously monitor extracellular pH (pH e ) and O 2 tension ( P O 2 ) of rainbow trout blood. In this closed system, adding CA to acidified, adrenergically stimulated RBCs short-circuited βNHE pH regulation, resulting in an increase in P O 2 by >30 mmHg, depending on the starting Hb-O 2 saturation and degree of initial acidification. Interestingly, in the absence of adrenergic stimulation, addition of CA still elevated P O 2 , albeit to a lesser extent, a response that was absent during general NHE inhibition. If plasma-accessible CA-mediated short-circuiting is operational in vivo , the combined Bohr-Root Effect system unique to teleost fishes could markedly enhance tissue O 2 delivery far in excess of that in vertebrates possessing a Bohr Effect alone and may lead to insights about the early evolution of the Root Effect.
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Use it or lose it? Sablefish, Anoplopoma fimbria, a species representing a fifth teleostean group where the βNHE associated with the red blood cell adrenergic stress response has been secondarily lost
Journal of Experimental Biology, 2010Co-Authors: Jodie L. Rummer, Mani Roshan-moniri, Shannon K. Balfry, Colin J. BraunerAbstract:Like most teleosts, sablefish (Anoplopoma fimbria Pallas 1814) blood exhibits a moderate Root Effect (~35% maximal desaturation), where a reduction in blood pH dramatically reduces O(2) carrying capacity, a mechanism important for oxygenating the eye and filling the swim bladder (SB) in teleosts. Although sablefish lack a SB, we observed a well-defined choroid rete at the eye. The adrenergically mediated cell swelling typically associated with a functional red blood cell (RBC) beta-adrenergic Na(+)/H(+) exchanger (betaNHE), which would normally protect RBC pH, and thus O(2) transport, during a generalized acidosis, was not observed in sablefish blood. Neither isoproterenol (a beta-agonist) nor 8-bromo cAMP could elicit this response. Furthermore, RBC osmotic shrinkage, known to stimulate NHEs in general and betaNHE in other teleosts such as trout and flounder, resulted in no significant regulatory volume increase (RVI), further supporting the absence of a functional RBC betaNHE. The onset of the Root Effect occurs at a much lower RBC pH (6.83-6.92) than in other teleosts, and thus RBC betaNHE may not be required to protect O(2) transport during a generalized acidosis in vivo. Phylogenetically, sablefish may represent a fifth group of teleosts exhibiting a secondary reduction or loss of betaNHE activity. However, sablefish have not lost the choroid rete at the eye (unlike in the other four groups), which may still function with the Root Effect to oxygenate the retina, but the low pH onset of the Root Effect may ensure haemoglobin (Hb)-O(2) binding is not compromised at the respiratory surface during a general acidosis in the absence of RBC betaNHE. The sablefish may represent an anomaly within the framework of Root Effect evolution, in that they possess a moderate Root Effect and a choroid rete at the eye, but lack the RBC betaNHE and the SB system.
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Use it or lose it? Sablefish, Anoplopoma fimbria, a species representing a fifth teleostean group where the betaNHE associated with the red blood cell adrenergic stress response has been secondarily lost.
The Journal of experimental biology, 2010Co-Authors: Jodie L. Rummer, Mani Roshan-moniri, Shannon K. Balfry, Colin J. BraunerAbstract:Like most teleosts, sablefish (Anoplopoma fimbria Pallas 1814) blood exhibits a moderate Root Effect (~35% maximal desaturation), where a reduction in blood pH dramatically reduces O(2) carrying capacity, a mechanism important for oxygenating the eye and filling the swim bladder (SB) in teleosts. Although sablefish lack a SB, we observed a well-defined choroid rete at the eye. The adrenergically mediated cell swelling typically associated with a functional red blood cell (RBC) beta-adrenergic Na(+)/H(+) exchanger (betaNHE), which would normally protect RBC pH, and thus O(2) transport, during a generalized acidosis, was not observed in sablefish blood. Neither isoproterenol (a beta-agonist) nor 8-bromo cAMP could elicit this response. Furthermore, RBC osmotic shrinkage, known to stimulate NHEs in general and betaNHE in other teleosts such as trout and flounder, resulted in no significant regulatory volume increase (RVI), further supporting the absence of a functional RBC betaNHE. The onset of the Root Effect occurs at a much lower RBC pH (6.83-6.92) than in other teleosts, and thus RBC betaNHE may not be required to protect O(2) transport during a generalized acidosis in vivo. Phylogenetically, sablefish may represent a fifth group of teleosts exhibiting a secondary reduction or loss of betaNHE activity. However, sablefish have not lost the choroid rete at the eye (unlike in the other four groups), which may still function with the Root Effect to oxygenate the retina, but the low pH onset of the Root Effect may ensure haemoglobin (Hb)-O(2) binding is not compromised at the respiratory surface during a general acidosis in the absence of RBC betaNHE. The sablefish may represent an anomaly within the framework of Root Effect evolution, in that they possess a moderate Root Effect and a choroid rete at the eye, but lack the RBC betaNHE and the SB system.
Guido Di Prisco - One of the best experts on this subject based on the ideXlab platform.
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Crystallization, preliminary X-ray diffraction studies and Raman microscopy of the major haemoglobin from the sub-Antarctic fish Eleginops maclovinus in the carbomonoxy form
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2010Co-Authors: Antonello Merlino, Daniela Coppola, Cinzia Verde, Daniela Giordano, Guido Di Prisco, Luigi Vitagliano, Anna Balsamo, Francesco P. Nicoletti, Barry D. Howes, Giulietta SmulevichAbstract:The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three\ud haemoglobins. The major haemoglobin (Hb1Em) displays the Root Effect, a\ud drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH.\ud The carbomonoxy form of HbEm1 has been crystallized in two different crystal\ud forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution\ud diffraction data have been collected for both forms (1.45 and 1.49 A ˚ resolution,\ud respectively). The high-frequency resonance Raman spectra collected from\ud the two crystal forms using excitation at 514 nm were almost indistinguishable.\ud Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural\ud characterization will shed light on the molecular mechanisms of cold adaptation\ud and the role of the Root Effect in fish haemoglobins
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An order-disorder transition plays a role in switching off the Root Effect in fish hemoglobins.
The Journal of biological chemistry, 2010Co-Authors: Alessandro Vergara, Cinzia Verde, Guido Di Prisco, Luigi Vitagliano, Antonello Merlino, Filomena Sica, Katia Marino, Lelio MazzarellaAbstract:Abstract The Root Effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-Effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α1β2 and α2β1 interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root Effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root Effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root Effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.
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Correlation between hemichrome stability and the Root Effect in tetrameric hemoglobins.
Biophysical journal, 2009Co-Authors: Alessandro Vergara, Cinzia Verde, Daniela Giordano, Guido Di Prisco, Marisa Franzese, Antonello Merlino, G. Bonomi, H. Caroline Lee, Jack Peisach, Lelio MazzarellaAbstract:Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root Effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-Effect Hb from T. bernacchii is also reported at 1.7 A resolution. This structure reveals a 5C state of the heme iron for both the alpha- and beta-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root Effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.
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The Root Effect – a structural and evolutionary perspective
Antarctic Science, 2007Co-Authors: Cinzia Verde, Alessandro Vergara, Daniela Giordano, Lelio Mazzarella, Guido Di PriscoAbstract:AbstractHaemoglobin carries oxygen from the environment to tissues; in vertebrates, it is contained in specialized cells, called erythrocytes. Over the last century, the study of the chemical properties of this haemoprotein has provided a wealth of information. One of its most important and ancient physiological features is the Root Effect, found in many teleost fish (and some amphibians). The Root Effect corresponds to an extreme pH sensitivity and can be described as an exaggerated Bohr Effect: it dictates to what extent the oxygen tension can be raised in acid-producing tissues. It is likely that the eye choroid rete represents the most ancient anatomical structure associated with the presence of Root Effect haemoglobins. This review describes our overall understanding of the molecular properties, biological occurrence, physiological role and evolutionary origin of Root Effect haemoglobins. The current knowledge of the structural properties of Root Effect haemoglobins is discussed in the light of recent results obtained on the haemoglobins of the coldadapted notothenioids Trematomus newnesi and T. bernacchii.
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High resolution crystal structure of deoxy hemoglobin from Trematomus bernacchii at different pH values: the role of histidine residues in modulating the strength of the Root Effect.
Proteins, 2006Co-Authors: Lelio Mazzarella, Cinzia Verde, Alessandro Vergara, Luigi Vitagliano, Antonello Merlino, G. Bonomi, Sonia Scala, Guido Di PriscoAbstract:The Root Effect is a widespread property in fish hemoglobins (Hbs) that produces a drastic reduction of cooperativity and oxygen-binding ability at acidic pH. Here, we report the high-resolution structure of the deoxy form of Hb isolated from the Antarctic fish Trematomus bernacchii (HbTb) crystallized at pH 6.2 and 8.4. The structure at acidic pH has been previously determined at a moderate resolution (Ito et al., J Mol Biol 1995;250:648-658). Our results provide a clear picture of the events occurring upon the pH increase from 6.2 to 8.4, observed within a practically unchanged crystal environment. In particular, at pH 8.4, the interaspartic hydrogen bond at the alpha(1)beta(2) interface is partially broken, suggesting a pK(a) close to 8.4 for Asp95alpha. In addition, a detailed survey of the histidine modifications, caused by the change in pH, also indicates that at least three hot regions of the molecule are modified (Ebeta helix, Cbeta-tail, CDalpha corner) and can be considered to be involved at various levels in the release of the Root protons. Most importantly, at the CDalpha corner, the break of the salt bridge Asp48alpha-His55alpha allows us to describe a detailed mechanism that transmits the modification from the CDalpha corner far to the alpha heme. More generally, the results shed light on the role played by the histidine residues in modulating the strength of the Root Effect and also support the emerging idea that the structural determinants, at least for a part of the Root Effect, are specific of each Hb endowed with this property.
Lelio Mazzarella - One of the best experts on this subject based on the ideXlab platform.
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Role of tertiary structures on the Root Effect in fish hemoglobins.
Biochimica et biophysica acta, 2013Co-Authors: Luca Ronda, Cinzia Verde, Lelio Mazzarella, Antonello Merlino, Stefano Bettati, Anna Balsamo, Andrea Mozzarelli, Alessandro VergaraAbstract:Abstract Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root Effect. Both tertiary and quaternary Effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH 6.2 and pH 8.4. At low pH ligation has negligible structural Effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T–R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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Protonation of histidine 55 affects the oxygen access to heme in the alpha chain of the hemoglobin from the Antarctic fish Trematomus bernacchii.
IUBMB life, 2011Co-Authors: Leonardo Boechi, Alessandro Vergara, Lelio Mazzarella, Filomena Sica, Marcelo A. Martí, Darío A. Estrin, Antonello MerlinoAbstract:The Root Effect describes the drastic drop of oxygen affinity and loss of cooperativity at acidic pH expressed in the hemoglobins (Hb) of certain fish. The comparison between the deoxy structures of the Root Effect Hb from the Antarctic fish Trematomus bernacchii (HbTb) at different pHs (pH = 6.2 and pH = 8.4) shows that the most significant differences are localized at the CDα region, where a salt bridge between Asp48 and His55 breaks during the low-to-high pH transition. In order to shed light on the relationship between pH, CDα loop structure and dynamics, and oxygen access to the active site in the alpha chain of HbTb, different computer simulation techniques were performed. Our results highlight the importance of the protonation of His55 in regulating oxygen access, underscoring its pivotal role in the structural and functional properties of HbTb. These data provide further support to the hypothesis that this residue might contribute to the release of Root protons in HbTb and underline the fact that an efficient transport of molecular oxygen in Hbs relies on a subtle balance of tertiary structure and protein conformational flexibility.
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An order-disorder transition plays a role in switching off the Root Effect in fish hemoglobins.
The Journal of biological chemistry, 2010Co-Authors: Alessandro Vergara, Cinzia Verde, Guido Di Prisco, Luigi Vitagliano, Antonello Merlino, Filomena Sica, Katia Marino, Lelio MazzarellaAbstract:Abstract The Root Effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-Effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α1β2 and α2β1 interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root Effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root Effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root Effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.
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Correlation between hemichrome stability and the Root Effect in tetrameric hemoglobins.
Biophysical journal, 2009Co-Authors: Alessandro Vergara, Cinzia Verde, Daniela Giordano, Guido Di Prisco, Marisa Franzese, Antonello Merlino, G. Bonomi, H. Caroline Lee, Jack Peisach, Lelio MazzarellaAbstract:Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root Effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-Effect Hb from T. bernacchii is also reported at 1.7 A resolution. This structure reveals a 5C state of the heme iron for both the alpha- and beta-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root Effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.
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The Root Effect – a structural and evolutionary perspective
Antarctic Science, 2007Co-Authors: Cinzia Verde, Alessandro Vergara, Daniela Giordano, Lelio Mazzarella, Guido Di PriscoAbstract:AbstractHaemoglobin carries oxygen from the environment to tissues; in vertebrates, it is contained in specialized cells, called erythrocytes. Over the last century, the study of the chemical properties of this haemoprotein has provided a wealth of information. One of its most important and ancient physiological features is the Root Effect, found in many teleost fish (and some amphibians). The Root Effect corresponds to an extreme pH sensitivity and can be described as an exaggerated Bohr Effect: it dictates to what extent the oxygen tension can be raised in acid-producing tissues. It is likely that the eye choroid rete represents the most ancient anatomical structure associated with the presence of Root Effect haemoglobins. This review describes our overall understanding of the molecular properties, biological occurrence, physiological role and evolutionary origin of Root Effect haemoglobins. The current knowledge of the structural properties of Root Effect haemoglobins is discussed in the light of recent results obtained on the haemoglobins of the coldadapted notothenioids Trematomus newnesi and T. bernacchii.
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Role of tertiary structures on the Root Effect in fish hemoglobins.
Biochimica et biophysica acta, 2013Co-Authors: Luca Ronda, Cinzia Verde, Lelio Mazzarella, Antonello Merlino, Stefano Bettati, Anna Balsamo, Andrea Mozzarelli, Alessandro VergaraAbstract:Abstract Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root Effect. Both tertiary and quaternary Effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH 6.2 and pH 8.4. At low pH ligation has negligible structural Effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T–R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.
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Crystallization, preliminary X-ray diffraction studies and Raman microscopy of the major haemoglobin from the sub-Antarctic fish Eleginops maclovinus in the carbomonoxy form
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2010Co-Authors: Antonello Merlino, Daniela Coppola, Cinzia Verde, Daniela Giordano, Guido Di Prisco, Luigi Vitagliano, Anna Balsamo, Francesco P. Nicoletti, Barry D. Howes, Giulietta SmulevichAbstract:The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three\ud haemoglobins. The major haemoglobin (Hb1Em) displays the Root Effect, a\ud drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH.\ud The carbomonoxy form of HbEm1 has been crystallized in two different crystal\ud forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution\ud diffraction data have been collected for both forms (1.45 and 1.49 A ˚ resolution,\ud respectively). The high-frequency resonance Raman spectra collected from\ud the two crystal forms using excitation at 514 nm were almost indistinguishable.\ud Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural\ud characterization will shed light on the molecular mechanisms of cold adaptation\ud and the role of the Root Effect in fish haemoglobins
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An order-disorder transition plays a role in switching off the Root Effect in fish hemoglobins.
The Journal of biological chemistry, 2010Co-Authors: Alessandro Vergara, Cinzia Verde, Guido Di Prisco, Luigi Vitagliano, Antonello Merlino, Filomena Sica, Katia Marino, Lelio MazzarellaAbstract:Abstract The Root Effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-Effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α1β2 and α2β1 interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root Effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root Effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root Effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.
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Correlation between hemichrome stability and the Root Effect in tetrameric hemoglobins.
Biophysical journal, 2009Co-Authors: Alessandro Vergara, Cinzia Verde, Daniela Giordano, Guido Di Prisco, Marisa Franzese, Antonello Merlino, G. Bonomi, H. Caroline Lee, Jack Peisach, Lelio MazzarellaAbstract:Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root Effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-Effect Hb from T. bernacchii is also reported at 1.7 A resolution. This structure reveals a 5C state of the heme iron for both the alpha- and beta-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root Effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.
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The Root Effect – a structural and evolutionary perspective
Antarctic Science, 2007Co-Authors: Cinzia Verde, Alessandro Vergara, Daniela Giordano, Lelio Mazzarella, Guido Di PriscoAbstract:AbstractHaemoglobin carries oxygen from the environment to tissues; in vertebrates, it is contained in specialized cells, called erythrocytes. Over the last century, the study of the chemical properties of this haemoprotein has provided a wealth of information. One of its most important and ancient physiological features is the Root Effect, found in many teleost fish (and some amphibians). The Root Effect corresponds to an extreme pH sensitivity and can be described as an exaggerated Bohr Effect: it dictates to what extent the oxygen tension can be raised in acid-producing tissues. It is likely that the eye choroid rete represents the most ancient anatomical structure associated with the presence of Root Effect haemoglobins. This review describes our overall understanding of the molecular properties, biological occurrence, physiological role and evolutionary origin of Root Effect haemoglobins. The current knowledge of the structural properties of Root Effect haemoglobins is discussed in the light of recent results obtained on the haemoglobins of the coldadapted notothenioids Trematomus newnesi and T. bernacchii.
