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.

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.

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.

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.

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.

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.

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.

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.

Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose.

During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the high-flying bar-headed goose (Anser indicus), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. To test the effects of observed substitutions on evolutionarily relevant genetic backgrounds, we synthesized all possible genotypic intermediates in the line of descent connecting the wildtype bar-headed goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed one major-effect mutation that significantly increased Hb-O2 affinity on all possible genetic backgrounds. Two other mutations exhibited smaller average effect sizes and less additivity across backgrounds. One of the latter mutations produced a concomitant increase in the autoxidation rate, a deleterious side-effect that was fully compensated by a second-site mutation at a spatially proximal residue. The experiments revealed three key insights: (i) subtle, localized structural changes can produce large functional effects; (ii) relative effect sizes of function-altering mutations may depend on the sequential order in which they occur; and (iii) compensation of deleterious pleiotropic effects may play an important role in the adaptive evolution of protein function.

Divergent and parallel routes of biochemical adaptation in high-altitude passerine birds from the Qinghai-Tibet Plateau.

When different species experience similar selection pressures, the probability of evolving similar adaptive solutions may be influenced by legacies of evolutionary history, such as lineage-specific changes in genetic background. Here we test for adaptive convergence in hemoglobin (Hb) function among high-altitude passerine birds that are native to the Qinghai-Tibet Plateau, and we examine whether convergent increases in Hb-O2 affinity have a similar molecular basis in different species. We documented that high-altitude parid and aegithalid species from the Qinghai-Tibet Plateau have evolved derived increases in Hb-O2 affinity in comparison with their closest lowland relatives in East Asia. However, convergent increases in Hb-O2 affinity and convergence in underlying functional mechanisms were seldom attributable to the same amino acid substitutions in different species. Using ancestral protein resurrection and site-directed mutagenesis, we experimentally confirmed two cases in which parallel substitutions contributed to convergent increases in Hb-O2 affinity in codistributed high-altitude species. In one case involving the ground tit (Parus humilis) and gray-crested tit (Lophophanes dichrous), parallel amino acid replacements with affinity-enhancing effects were attributable to nonsynonymous substitutions at a CpG dinucleotide, suggesting a possible role for mutation bias in promoting recurrent changes at the same site. Overall, most altitude-related changes in Hb function were caused by divergent amino acid substitutions, and a select few were caused by parallel substitutions that produced similar phenotypic effects on the divergent genetic backgrounds of different species.

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.

Emergence of a Chimeric Globin Pseudogene and Increased Hemoglobin Oxygen Affinity Underlie the Evolution of Aquatic Specializations in Sirenia.

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller's sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb-O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb-O2 affinity in (sub)Arctic Steller's sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

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.

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.

Oxygenation properties of hemoglobin and the evolutionary origins of isoform multiplicity in an amphibious air-breathing fish, the blue-spotted mudskipper (Boleophthalmus pectinirostris).

Among the numerous lineages of teleost fish that have independently transitioned from obligate water breathing to facultative air breathing, evolved properties of hemoglobin (Hb)-O2 transport may have been shaped by the prevalence and severity of aquatic hypoxia (which influences the extent to which fish are compelled to switch to aerial respiration) as well as the anatomical design of air-breathing structures and the cardiovascular system. Here, we examined the structure and function of Hbs in an amphibious, facultative air-breathing fish, the blue-spotted mudskipper (Boleophthalmus pectinirostris). We also characterized the genomic organization of the globin gene clusters of the species and we integrated phylogenetic and comparative genomic analyses to unravel the duplicative history of the genes that encode the subunits of structurally distinct mudskipper Hb isoforms (isoHbs). The B. pectinirostris isoHbs exhibit high intrinsic O2 affinities, similar to those of hypoxia-tolerant, water-breathing teleosts, and remarkably large Bohr effects. Genomic analysis of conserved synteny revealed that the genes that encode the α-type subunits of the two main adult isoHbs are members of paralogous gene clusters that represent products of the teleost-specific whole-genome duplication. Experiments revealed no appreciable difference in the oxygenation properties of co-expressed isoHbs in spite of extensive amino acid divergence between the alternative α-chain subunit isoforms. It therefore appears that the ability to switch between aquatic and aerial respiration does not necessarily require a division of labor between functionally distinct isoHbs with specialized oxygenation properties.

The Zebrafish Cytochrome b5/Cytochrome b5 Reductase/NADH System Efficiently Reduces Cytoglobins 1 and 2: Conserved Activity of Cytochrome b5/Cytochrome b5 Reductases during Vertebrate Evolution.

Cytoglobin is a heme protein evolutionarily related to hemoglobin and myoglobin. Cytoglobin is expressed ubiquitously in mammalian tissues; however, its physiological functions are yet unclear. Phylogenetic analyses indicate that the cytoglobin gene is highly conserved in vertebrate clades, from fish to reptiles, amphibians, birds, and mammals. Most proposed roles for cytoglobin require the maintenance of a pool of reduced cytoglobin (FeII). We have shown previously that the human cytochrome b5/cytochrome b5 reductase system, considered a quintessential hemoglobin/myoglobin reductant, can reduce human and zebrafish cytoglobins ≤250-fold faster than human hemoglobin or myoglobin. It was unclear whether this reduction of zebrafish cytoglobins by mammalian proteins indicates a conserved pathway through vertebrate evolution. Here, we report the reduction of zebrafish cytoglobins 1 and 2 by the zebrafish cytochrome b5 reductase and the two zebrafish cytochrome b5 isoforms. In addition, the reducing system also supports reduction of Globin X, a conserved globin in fish and amphibians. Indeed, the zebrafish reducing system can maintain a fully reduced pool for both cytoglobins, and both cytochrome b5 isoforms can support this process. We determined the P50 for oxygen to be 0.5 Torr for cytoglobin 1 and 4.4 Torr for cytoglobin 2 at 25 °C. Thus, even at low oxygen tensions, the reduced cytoglobins may exist in a predominant oxygen-bound form. Under these conditions, the cytochrome b5/cytochrome b5 reductase system can support a conserved role for cytoglobins through evolution, providing electrons for redox signaling reactions such as nitric oxide dioxygenation, nitrite reduction, and phospholipid oxidation.

Tissue-dependent variation of hydrogen sulfide homeostasis in anoxic freshwater turtles.

Hydrogen sulfide (H2S) controls numerous physiological responses. To understand its proposed role in metabolic suppression, we measured free H2S and bound sulfane sulfur (BSS) in tissues of the freshwater turtle Trachemys scriptaelegans, a species undergoing strong metabolic suppression when cold and anoxic. In warm normoxic turtles, free H2S was higher in red blood cells (RBCs) and kidney (∼9-10 µmol l-1) than in brain, liver and lung (∼1-2 µmol l-1). These values overall aligned with the tissue H2S-generating enzymatic activity. BSS levels were similar in all tissues (∼0.5 µmol l-1) but ∼100-fold higher in RBCs, which have a high thiol content, suggesting that RBCs function as a circulating H2S reservoir. Cold acclimation caused significant changes in free and bound H2S in liver, brain and RBCs, but anoxia had no further effect, except in the brain. These results show tissue-dependent sulfide signaling with a potential role in brain metabolic suppression during anoxia in turtles.

Turtles maintain mitochondrial integrity but reduce mitochondrial respiratory capacity in the heart after cold acclimation and anoxia.

Mitochondria are important to cellular homeostasis, but can become a dangerous liability when cells recover from hypoxia. Anoxia-tolerant freshwater turtles show reduced mitochondrial respiratory capacity and production of reactive oxygen species (ROS) after prolonged anoxia, but the mechanisms are unclear. Here, we investigated whether this mitochondrial suppression originates from downregulation of mitochondrial content or intrinsic activity by comparing heart mitochondria from (1) warm (25°C) normoxic, (2) cold-acclimated (4°C) normoxic and (3) cold-acclimated anoxic turtles. Transmission electron microscopy of heart ventricle revealed that these treatments did not affect mitochondrial volume density and morphology. Furthermore, neither enzyme activity, protein content nor supercomplex distribution of electron transport chain (ETC) enzymes changed significantly. Instead, our data imply that turtles inhibit mitochondrial respiration rate and ROS production by a cumulative effect of slight inhibition of ETC complexes. Together, these results show that maintaining mitochondrial integrity while inhibiting overall enzyme activities are important aspects of anoxia tolerance.