Generation and validation of a myoglobin knockout zebrafish model.

Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.

Evolution of an extreme hemoglobin phenotype contributed to the sub-Arctic specialization of extinct Steller's sea cows.

The extinct Steller's sea cow (Hydrodamalis gigas; †1768) was a whale-sized marine mammal that manifested profound morphological specializations to exploit the harsh coastal climate of the North Pacific. Yet despite first-hand accounts of their biology, little is known regarding the physiological adjustments underlying their evolution to this environment. Here, the adult-expressed hemoglobin (Hb; α2ß/δ2) of this sirenian is shown to harbor a fixed amino acid replacement at an otherwise invariant position (ß/δ82Lys→Asn) that alters multiple aspects of Hb function. First, our functional characterization of recombinant sirenian Hb proteins demonstrates that the Hb-O2 affinity of this sub-Arctic species was less affected by temperature than those of living (sub)tropical sea cows. This phenotype presumably safeguarded O2 delivery to cool peripheral tissues and largely arises from a reduced intrinsic temperature sensitivity of the H. gigas protein. Additional experiments on H. gigas ß/δ82Asn→Lys mutant Hb further reveal this exchange renders Steller's sea cow Hb unresponsive to the potent intraerythrocytic allosteric effector 2,3-diphosphoglycerate, a radical modification that is the first documented example of this phenotype among mammals. Notably, ß/δ82Lys→Asn moreover underlies the secondary evolution of a reduced blood-O2 affinity phenotype that would have promoted heightened tissue and maternal/fetal O2 delivery. This conclusion is bolstered by analyses of two Steller's sea cow prenatal Hb proteins (Hb Gower I; ζ2ε2 and HbF; α2γ2) that suggest an exclusive embryonic stage expression pattern, and reveal uncommon replacements in H. gigas HbF (γ38Thr→Ile and γ101Glu→Asp) that increased Hb-O2 affinity relative to dugong HbF. Finally, the ß/δ82Lys→Asn replacement of the adult/fetal protein is shown to increase protein solubility, which may have elevated red blood cell Hb content within both the adult and fetal circulations and contributed to meeting the elevated metabolic (thermoregulatory) requirements and fetal growth rates associated with this species cold adaptation.

In 1741, shipwrecked naturalist Georg Wilhelm Steller made detailed observations of large marine mammals grazing on seaweed in the shallow waters surrounding a remote island in the North Pacific Ocean. Within thirty years, these 'Steller's sea cows' had been hunted to extinction. Unlike their remaining tropical relatives ­ dugongs and manatees ­ Steller's sea cows were specialized to cold, sub-Arctic environments. Measuring up to 10 meters long, they were much larger than other sea cow species. This, along with having very thick skin, helped them to reduce heat loss. Previous work showed that the hemoglobin protein ­ which binds to and carries oxygen around mammalian bodies ­ of Steller's sea cows had a decreased affinity for oxygen, resulting in greater delivery of oxygen to organs and tissues. It was thought that this could be an adaptation to fuel heightened metabolic heat production in cold conditions. Studies of ancient DNA also identified the substitution of a single building block in the Steller's sea cow hemoglobin protein that is not present in other mammals and was suspected to underlie this modification. To determine how this unique substitution affects Steller's sea cow hemoglobin function ­ and whether it contributed to their ability to live in cold environments ­ Signore et al. generated hemoglobin proteins of Steller's sea cows, dugongs and Florida manatees. Testing their biochemical properties showed that this single exchange profoundly alters multiple aspects of how the Steller's sea cow hemoglobin works. Alongside reducing hemoglobin's oxygen affinity, the Steller's sea cow substitution also makes the protein more soluble, potentially increasing the level of hemoglobin within red blood cells. Additionally, it eliminates hemoglobin sensitivity to a molecule involved in oxygen binding ­ known as DPG ­ saving energy by no longer requiring production of this molecule. Furthermore, the same substitution makes hemoglobin less sensitive to changes in temperature, which would have helped to safeguard the delivery of oxygen to cool limbs and other extremities, reducing costly heat loss. Together, these changes in hemoglobin would have helped the Steller's sea cow to more efficiently transport oxygen around the body. Importantly, generating and testing Steller's sea cow pre-natal hemoglobins suggested this substitution may have also helped to enhance the fetal growth rate of these immense marine mammals by improving gas exchange between the mother and fetus. Signore et al. have revealed how a mutated form of hemoglobin allowed an extinct mammal to adapt to an extreme environment. Similar methods could be used to understand the physiological attributes of other extinct animals. In the future, this increased understanding of hemoglobin mutations could aid the development of human hemoglobin substitutes for therapeutic uses.

Low production of mitochondrial reactive oxygen species after anoxia and reoxygenation in turtle hearts.

Extremely anoxia-tolerant animals, such as freshwater turtles, survive anoxia and reoxygenation without sustaining tissue damage to their hearts. In contrast, for mammals, the ischemia-reperfusion (IR) injury that leads to tissue damage during a heart attack is initiated by a burst of superoxide (O2·-) production from the mitochondrial respiratory chain upon reperfusion of ischemic tissue. Whether turtles avoid oxidative tissue damage because of an absence of mitochondrial superoxide production upon reoxygenation, or because the turtle heart is particularly protected against this damage, is unclear. Here, we investigated whether there was an increase in mitochondrial O2·- production upon the reoxygenation of anoxic red-eared slider turtle hearts in vivo and in vitro. This was done by measuring the production of H2O2, the dismutation product of O2·-, using the mitochondria-targeted mass-spectrometric probe in vivo MitoB, while in parallel assessing changes in the metabolites driving mitochondrial O2·- production, succinate, ATP and ADP levels during anoxia, and H2O2 consumption and production rates of isolated heart mitochondria. We found that there was no excess production of in vivo H2O2 during 1 h of reoxygenation in turtles after 3 h anoxia at room temperature, suggesting that turtle hearts most likely do not suffer oxidative injury after anoxia because their mitochondria produce no excess O2·- upon reoxygenation. Instead, our data support the conclusion that both the low levels of succinate accumulation and the maintenance of ADP levels in the anoxic turtle heart are key factors in preventing the surge of O2·- production upon reoxygenation.

Structural and oxidative investigation of a recombinant high-yielding fetal hemoglobin mutant.

Human fetal hemoglobin (HbF) is an attractive starting protein for developing an effective agent for oxygen therapeutics applications. This requires that HbF can be produced in heterologous systems at high levels and in a homogeneous form. The introduction of negative charges on the surface of the α-chain in HbF can enhance the recombinant production yield of a functional protein in Escherichia coli. In this study, we characterized the structural, biophysical, and biological properties of an HbF mutant carrying four additional negative charges on each α-chain (rHbFα4). The 3D structure of the rHbFα4 mutant was solved with X-ray crystallography at 1.6 Å resolution. Apart from enabling a higher yield in recombinant protein production in E. coli, we observed that the normal DNA cleavage activity of the HbF was significantly lowered, with a four-time reduced rate constant for the rHbFα4 mutant. The oxygen-binding properties of the rHbFα4 mutant were identical to the wild-type protein. No significant difference between the wild-type and rHbFα4 was observed for the investigated oxidation rates (autoxidation and H2O2-mediated ferryl formation). However, the ferryl reduction reaction indicated some differences, which appear to be related to the reaction rates linked to the α-chain.

Evolution and molecular basis of a novel allosteric property of crocodilian hemoglobin.

The extraordinary breath-hold diving capacity of crocodilians has been ascribed to a unique mode of allosterically regulating hemoglobin (Hb)-oxygenation in circulating red blood cells. We investigated the origin and mechanistic basis of this novel biochemical phenomenon by performing directed mutagenesis experiments on resurrected ancestral Hbs. Comparisons of Hb function between the common ancestor of archosaurs (the group that includes crocodilians and birds) and the last common ancestor of modern crocodilians revealed that regulation of Hb-O2 affinity via allosteric binding of bicarbonate ions represents a croc-specific innovation that evolved in combination with the loss of allosteric regulation by ATP binding. Mutagenesis experiments revealed that evolution of the novel allosteric function in crocodilians and the concomitant loss of ancestral function were not mechanistically coupled and were caused by different sets of substitutions. The gain of bicarbonate sensitivity in crocodilian Hb involved the direct effect of few amino acid substitutions at key sites in combination with indirect effects of numerous other substitutions at structurally disparate sites. Such indirect interaction effects suggest that evolution of the novel protein function was conditional on neutral mutations that produced no adaptive benefit when they first arose but that contributed to a permissive background for subsequent function-altering mutations at other sites. Due to the context dependence of causative substitutions, the unique allosteric properties of crocodilian Hb cannot be easily transplanted into divergent homologs of other species.

New insights into survival strategies to oxygen deprivation in anoxia-tolerant vertebrates.

Hypoxic environments pose a severe challenge to vertebrates and even short periods of oxygen deprivation are often lethal as they constrain aerobic ATP production. However, a few ectotherm vertebrates are capable of surviving long-term hypoxia or even anoxia with little or no damage. Among these, freshwater turtles and crucian carp are the recognized champions of anoxia tolerance, capable of overwintering in complete oxygen deprivation for months at freezing temperatures by entering a stable hypometabolic state. While all steps of the oxygen cascade are adjusted in response to oxygen deprivation, this review draws from knowledge of freshwater turtles and crucian carp to highlight mechanisms regulating two of these steps, namely oxygen transport in the blood and oxygen utilization in mitochondria during three sequential phases: before anoxia, when hypoxia develops, during anoxia, and after anoxia at reoxygenation. In cold hypoxia, reduced red blood cell concentration of ATP plays a crucial role in increasing blood oxygen affinity and/or reducing oxygen unloading to tissues, to adjust aerobic metabolism to decrease ambient oxygen. In anoxia, metabolic rewiring of oxygen utilization keeps largely unaltered NADH/NAD+ ratios and limits ADP degradation and succinate buildup. These critical adjustments make it possible to restart mitochondrial respiration and energy production with little generation of reactive oxygen species at reoxygenation when oxygen is again available. Inhibition of key metabolic enzymes seems to play crucial roles in these responses, in particular mitochondrial complex V, although identifying the nature of such inhibition(s) in vivo remains a challenge for future studies.

Ontogeny of hemoglobin­oxygen binding and multiplicity in the obligate air-breathing fish Arapaima gigas.

The evolutionary and ontogenetic changes from water- to air-breathing result in major changes in the cardiorespiratory systems. However, the potential changes in hemoglobin's (Hb) oxygen binding properties during ontogenetic transitions to air-breathing remain poorly understood. Here we investigated Hb multiplicity and O2 binding in hemolysates and Hb components from juveniles and adults of the obligate air-breathing pirarucu (Arapaima gigas) that starts life as water-breathing hatchlings. Contrasting with previous electrophoresis studies that report one or two isoHbs in adults, isoelectric focusing (IEF) resolved the hemolysates from both stages into four major bands, which exhibited identical O2 binding properties (i.e. O2 affinities, cooperativity coefficients, and sensitivities to pH and the major organic phosphate effectors), also as compared to the cofactor-free hemolysates. Of note, the multiplicity pattern recurred upon reanalyses of the most-abundant fractions isolated from the juvenile and the adult stages, suggesting possible stabilization of different quaternary states with different isoelectric points during the purification procedure. The study demonstrates unchanged Hb-O2 binding properties during development, despite the pronounced differences in O2 availability between the two media, which harmonizes with findings based on a broader spectrum of interspecific comparisons. Taken together, these results disclose that obligate air-breathing in Arapaima is not contingent upon changes in Hb multiplicity and O2 binding characteristics.

Genetic variation in haemoglobin is associated with evolved changes in breathing in high-altitude deer mice.

Physiological systems often have emergent properties but the effects of genetic variation on physiology are often unknown, which presents a major challenge to understanding the mechanisms of phenotypic evolution. We investigated whether genetic variants in haemoglobin (Hb) that contribute to high-altitude adaptation in deer mice (Peromyscus maniculatus) are associated with evolved changes in the control of breathing. We created F2 inter-population hybrids of highland and lowland deer mice to test for phenotypic associations of α- and ß-globin variants on a mixed genetic background. Hb genotype had expected effects on Hb-O2 affinity that were associated with differences in arterial O2 saturation in hypoxia. However, high-altitude genotypes were also associated with breathing phenotypes that should contribute to enhancing O2 uptake in hypoxia. Mice with highland α-globin exhibited a more effective breathing pattern, with highland homozygotes breathing deeper but less frequently across a range of inspired O2, and this difference was comparable to the evolved changes in breathing pattern in deer mouse populations native to high altitude. The ventilatory response to hypoxia was augmented in mice that were homozygous for highland ß-globin. The association of globin variants with variation in breathing phenotypes could not be recapitulated by acute manipulation of Hb-O2 affinity, because treatment with efaproxiral (a synthetic drug that acutely reduces Hb-O2 affinity) had no effect on breathing in normoxia or hypoxia. Therefore, adaptive variation in Hb may have unexpected effects on physiology in addition to the canonical function of this protein in circulatory O2 transport.

Changes in hemoglobin function and isoform expression during embryonic development in the American alligator, Alligator mississippiensis.

In the developing embryos of egg-laying vertebrates, O2 flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O2 flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O2 transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O2). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O2 affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O2 affinity and high CO2 sensitivity. These results indicate that whole blood O2 affinity is mainly regulated by ATP in the early embryo and by CO2 and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

Sulfide metabolism and the mechanism of torpor.

Hibernation is a powerful response of a number of mammalian species to reduce energy during the cold winter season, when food is scarce. Mammalian hibernators survive winter by spending most of the time in a state of torpor, where basal metabolic rate is strongly suppressed and body temperature comes closer to ambient temperature. These torpor bouts are regularly interrupted by short arousals, where metabolic rate and body temperature spontaneously return to normal levels. The mechanisms underlying these changes, and in particular the strong metabolic suppression of torpor, have long remained elusive. As summarized in this Commentary, increasing evidence points to a potential key role for hydrogen sulfide (H2S) in the suppression of mitochondrial respiration during torpor. The idea that H2S could be involved in hibernation originated in some early studies, where exogenous H2S gas was found to induce a torpor-like state in mice, and despite some controversy, the idea persisted. H2S is a widespread signaling molecule capable of inhibiting mitochondrial respiration in vitro and studies found significant in vivo changes in endogenous H2S metabolites associated with hibernation or torpor. Along with increased expression of H2S-synthesizing enzymes during torpor, H2S degradation catalyzed by the mitochondrial sulfide:quinone oxidoreductase (SQR) appears to have a key role in controlling H2S availability for inhibiting respiration. Specifically, in thirteen-lined squirrels, SQR is highly expressed and inhibited in torpor, possibly by acetylation, thereby limiting H2S oxidation and causing inhibition of respiration. H2S may also control other aspects associated with hibernation, such as synthesis of antioxidant enzymes and of SQR itself.

New insights into the allosteric effects of CO2 and bicarbonate on crocodilian hemoglobin.

Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O2 binding is allosterically regulated by bicarbonate, which forms in red blood cells upon hydration of CO2. Although known for decades, this remarkable mode of allosteric control has not yet been experimentally verified with direct evidence of bicarbonate binding to crocodilian Hb, probably because of confounding CO2-mediated effects. Here, we provide the first quantitative analysis of the separate allosteric effects of CO2 and bicarbonate on purified Hb of the spectacled caiman (Caiman crocodilus). Using thin-layer gas diffusion chamber and Tucker chamber techniques, we demonstrate that both CO2 and bicarbonate bind to Hb with high affinity and strongly decrease O2 saturation of Hb. We propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O2 affinity T conformation of Hb. These results provide the first experimental evidence that bicarbonate binds directly to crocodilian Hb and promotes O2 delivery independently of CO2. Using the gas diffusion chamber, we observed similar effects in Hbs of a phylogenetically diverse set of other caiman, alligator and crocodile species, suggesting that the unique mode of allosteric regulation by CO2 and bicarbonate evolved >80-100 million years ago in the common ancestor of crocodilians. Our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where the build-up of erytrocytic CO2 and bicarbonate ions during breath-hold diving or digestion facilitates O2 delivery, while Hb desaturation facilitates CO2 transport as protein-bound CO2 and bicarbonate.

Evolution of hemoglobin function in tropical air-breathing catfishes.

The evolution of hemoglobin function in the transition from water- to air-breathing has been highly debated but remains unresolved. Here, we characterized the hemoglobin function in five closely related water- and air-breathing catfishes. We identify distinct directions of hemoglobin evolution in the clades that evolved air-breathing, and we show strong selection on hemoglobin function within the catfishes. These findings show that the lack of a general direction in hemoglobin function in the transition from water- to air-breathing may have resulted from divergent selection on hemoglobin function in independent clades of air-breathing fishes.

Suppression of mitochondrial respiration by hydrogen sulfide in hibernating 13-lined ground squirrels.

Hibernating mammals may suppress their basal metabolic rate during torpor by up to 95% to reduce energy expenditure during winter, but the underlying mechanisms remain poorly understood. Here we show that hydrogen sulfide (H2S), a ubiquitous signaling molecule, is a powerful inhibitor of respiration of liver mitochondria isolated from torpid 13-lined ground squirrels, but has a weak effect on mitochondria isolated during summer and hibernation arousals, where metabolic rate is normal. Consistent with these in vitro effects, we find strong seasonal variations of in vivo levels of H2S in plasma and increases of H2S levels in the liver of squirrels during torpor compared to levels during arousal and summer. The in vivo changes of liver H2S levels correspond with low activity of the mitochondrial H2S oxidizing enzyme sulfide:quinone oxidoreductase (SQR) during torpor. Taken together, these results suggest that during torpor, H2S accumulates in the liver due to a low SQR activity and contributes to inhibition of mitochondrial respiration, while during arousals and summer these effects are reversed, H2S is degraded by active SQR and mitochondrial respiration rates increase. This study provides novel insights into mechanisms underlying mammalian hibernation, pointing to SQR as a key enzyme involved in the control of mitochondrial function.

High temperature impairs mitochondrial function in rainbow trout cardiac mitochondria.

Mitochondria provide cellular energy through oxidative phosphorylation, and thus temperature-induced constraints on mitochondrial function may be crucial to animal aerobic scope and thermal tolerance. Here, we report the effect of temperature in the range 5-30°C on respiration rates of isolated cardiac mitochondria from rainbow trout (Oncorhynchus mykiss) studied by high-resolution respirometry and spectrophotometric enzyme activity assays. Arrhenius breakpoint temperature analysis indicated that mitochondrial respiration rates under phosphorylating and fully uncoupled conditions increased exponentially up to 20°C, but stopped increasing at higher temperatures. In contrast, respiration rates measured under non-phosphorylating leak conditions continued to increase up to 30°C. The decrease in the ratio between phosphorylating and uncoupled respiration at high temperature indicated that phosphorylation was gradually impaired with increasing temperature, possibly because of the steadily increasing proton leak across the membrane. In addition, we found that complex I (NADH dehydrogenase) activity decreased above 20°C, similarly to mitochondrial respiration, and that complex I was unstable in the presence of detergents, suggesting that it may be particularly sensitive to changes in its interaction with membrane phospholipids. In contrast, complex II (succinate dehydrogenase) maintained activity at temperatures above 20°C, although succinate oxidation was insufficient to compensate for the loss of complex I activity in intact mitochondria. Together, these results indicate that the temperature-induced decrease in cardiac mitochondrial function coincides with the temperature at which trout aerobic scope peaks, and is largely due to impaired phosphorylation and complex I activity.

Carbon dioxide and bicarbonate accumulation in caiman erythrocytes during diving.

The ability of crocodilian haemoglobins to bind HCO3 - has been appreciated for more than half a century, but the functional implication of this is exceptional mechanism has not previously been assessed in vivo Therefore, the goal of the present study was to address the hypothesis that CO2 primarily binds to Hb, rather than being accumulated in plasma as in other vertebrates, during diving in caimans. Here, we demonstrate that CO2 primarily accumulates within the erythrocyte during diving and that most of the accumulated CO2 is bound to haemoglobin. Furthermore, we show that this HCO3 --binding is tightly associated with the progressive blood deoxygenation during diving, therefore, crocodilians differ from the classic vertebrate pattern, where HCO3 - accumulates in the plasma upon excretion from the erythrocytes by the Cl--HCO3 --exchanger.

Exploring pathways of NO and H2S signaling in metabolic depression: The case of anoxic turtles.

In contrast to most vertebrates, freshwater turtles of the genera Trachemys and Chrysemys survive total oxygen deprivation for long periods of time. This remarkable tolerance makes them ideal August Krogh's model animals to study adaptions to survive oxygen deprivation. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H2S) and their metabolic derivatives are central in regulating the physiological responses to oxygen deprivation. Here, we explore the role of these signaling molecules in the anoxia tolerance of the freshwater turtle, including metabolic suppression and protection against oxidative damage with oxygen deprivation. We describe the interaction of NO and H2S with protein thiols and specifically how this regulates the function of central metabolic enzymes. These interactions contribute both to metabolic suppression and to prevent oxidative damage with oxygen deprivation. Furthermore, NO and H2S interact with ferrous and ferric heme iron, respectively, which affects the activity of central heme proteins. In turtles, these interactions contribute to regulate oxygen consumption in the mitochondria, as well as vascular tone and blood flow during oxygen deprivation. The versatile biological effects of NO and H2S underscore the importance of these volatile signaling molecules in the remarkable tolerance of freshwater turtles to oxygen deprivation.

Effect of NH2-terminal acetylation on the oxygenation properties of vertebrate haemoglobin.

In vertebrate haemoglobin (Hb), the NH2-terminal residues of the α- and ß-chain subunits are thought to play an important role in the allosteric binding of protons (Bohr effect), CO2 (as carbamino derivatives), chloride ions, and organic phosphates. Accordingly, acetylation of the α- and/or ß-chain NH2-termini may have significant effects on the oxygenation properties of Hb. Here we investigate the effect of NH2-terminal acetylation by using a newly developed expression plasmid system that enables us to compare recombinantly expressed Hbs that are structurally identical except for the presence or absence of NH2-terminal acetyl groups. Experiments with native and recombinant Hbs of representative vertebrates reveal that NH2-terminal acetylation does not impair the Bohr effect, nor does it significantly diminish responsiveness to allosteric cofactors, such as chloride ions or organic phosphates. These results suggest that observed variation in the oxygenation properties of vertebrate Hbs is principally explained by amino acid divergence in the constituent globin chains rather than post-translational modifications of the globin chain NH2-termini.

Stable mitochondrial CICIII2 supercomplex interactions in reptiles versus homeothermic vertebrates.

The association of complex I (CI), complex III (CIII) and complex IV (CIV) of the mitochondrial electron transport chain into stable high molecular weight supercomplexes (SCs) has been observed in several prokaryotes and eukaryotes, but among vertebrates it has only been examined in mammals. The biological role of these SCs is unclear but suggestions so far include enhanced electron transfer between complexes, decreased production of the reactive oxygen species (ROS) O2- and H2O2, or enhanced structural stability. Here, we provide the first overview on the stability, composition and activity of mitochondrial SCs in representative species of several vertebrate classes to determine patterns of SC variation across endotherms and ectotherms. We found that the stability of the CICIII2 SC and the inclusion of CIV within the SC varied considerably. Specifically, when solubilized by the detergent DDM, mitochondrial CICIII2 SCs were unstable in endotherms (birds and mammals) and highly stable in reptiles. Using mass-spectrometric complexomics, we confirmed that the CICIII2 is the major SC in the turtle, and that 90% of CI is found in this highly stable SC. Interestingly, the presence of stable SCs did not prevent mitochondrial H2O2 production and was not associated with elevated respiration rates of mitochondria isolated from the examined species. Together, these data show that SC stability varies among vertebrates and is greatest in poikilothermic reptiles and weakest in endotherms. This pattern suggests an adaptive role of SCs to varying body temperature, but not necessarily a direct effect on electron transfer or in the prevention of ROS production.

Structure and function of crocodilian hemoglobins and allosteric regulation by chloride, ATP, and CO2.

Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO2. Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and ß-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO2, which would permit effective O2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O2-affinities, there is considerable variation in sensitivity to Cl- ions and ATP, which appears to be at least partly attributable to variation in the extent of NH2-terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.