Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent two-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Neuroglobin, recently discovered in the brain and in the retina of vertebrates, belongs to the class of hexacoordinate globins, in which the distal histidine coordinates the iron center in both the Fe(II) and Fe(III) forms. As for most other hexacoordinate globins, the physiological function of neuroglobin is still unclear, but seems to be related to neuronal survival following acute hypoxia. In this study, we have addressed the question whether human neuroglobin could act as a scavenger of toxic species, such as nitrogen monoxide, peroxynitrite, and hydrogen peroxide, which are generated at high levels in the brain during hypoxia; we have also investigated the kinetics of the reactions of its Fe(III) (metNGB) and Fe(II)NO forms with several reagents. Binding of cyanide or NO ⅐ to metNGB follows bi-exponential kinetics, showing the existence of two different protein conformations. In the presence of excess NO ⅐ , metNGB is converted into NGBFe(II)NO by reductive nitrosylation, in analogy to the reactions of NO ⅐ with metmyoglobin and methemoglobin. The Fe(II)NO form of neuroglobin is oxidized to metNGB by peroxynitrite and dioxygen, two reactions that also take place in hemoglobin, albeit at lower rates. In contrast to myoglobin and hemoglobin, metNGB unexpectedly does not generate the cytotoxic ferryl form of the protein upon addition of either peroxynitrite or hydrogen peroxide. Taken together, our data indicate that human neuroglobin may be an efficient scavenger of reactive oxidizing species and thus may play a role in the cellular defense against oxidative stress.

To investigate the predictability of genetic adaptation, we examined the molecular basis of convergence in hemoglobin function in comparisons involving 56 avian taxa that have contrasting altitudinal range limits. Convergent increases in hemoglobin-oxygen affinity were pervasive among high-altitude taxa, but few such changes were attributable to parallel amino acid substitutions at key residues. Thus, predictable changes in biochemical phenotype do not have a predictable molecular basis. Experiments involving resurrected ancestral proteins revealed that historical substitutions have context-dependent effects, indicating that possible adaptive solutions are contingent on prior history. Mutations that produce an adaptive change in one species may represent precluded possibilities in other species because of differences in genetic background.

Two new globin proteins have recently been discovered in vertebrates, neuroglobin in neurons and cytoglobin in all tissues, both showing heme hexacoordination by the distal His(E7) in the absence of gaseous ligands. In analogy to hemoglobin and myoglobin, neuroglobin and cytoglobin are supposedly involved in O 2 storage and delivery, although their physiological role remains to be solved. Here we report O 2 equilibria of recombinant human neuroglobin (NGB) and cytoglobin (CYGB) measured under close to physiological conditions and at varying temperature and pH ranges. NGB shows both alkaline and acid Bohr effects (pH-dependent O 2 affinity) and temperature-dependent enthalpy of oxygenation. O 2 and CO binding equilibrium studies on neuroglobin mutants strongly suggest that the bound O 2 is stabilized by interactions with His(E7) and that this residue functions as a major Bohr group in the presence of Lys(E10). As shown by the titration of free thiols with 4,4 -dithiodipyridine and by mass spectrometry, this mechanism of modulating O 2 affinity is independent of formation of an internal disulfide bond under the experimental conditions used, which stabilize thiols in the reduced form. In CYGB, O 2 binding is cooperative, consistent with its proposed dimeric structure. Similar to myoglobin but in contrast to NGB, O 2 binding to CYGB is pH-independent and exothermic throughout the temperature range investigated. Our data support the hypothesis that CYGB may be involved in O 2 -requiring metabolic processes. In contrast, the lower O 2 affinity in NGB does not appear compatible with a physiological role involving mitochondrial O 2 supply at the low O 2 tensions found within neurons.

Adaptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. Here we investigate these possibilities by conducting a combined evolutionary and functional analysis of duplicated globin genes in natural populations of deer mice (Peromyscus maniculatus) that are adapted to different elevational zones. A multilocus analysis of nucleotide polymorphism and linkage disequilibrium revealed that high-altitude adaptation of deer mouse hemoglobin involves parallel functional differentiation at multiple unlinked gene duplicates: two ␣-globin paralogs on chromosome 8 and two ␤-globin paralogs on chromosome 1. Differences in O 2-binding affinity of the alternative ␤-chain hemoglobin isoforms were entirely attributable to allelic differences in sensitivity to 2,3-diphosphoglycerate (DPG), an allosteric cofactor that stabilizes the low-affinity, deoxygenated conformation of the hemoglobin tetramer. The two-locus ␤-globin haplotype that predominates at high altitude is associated with suppressed DPGsensitivity (and hence, increased hemoglobin-O 2 affinity), which enhances pulmonary O2 loading under hypoxia. The discovery that allelic differences in DPG-sensitivity contribute to adaptive variation in hemoglobin-O 2 affinity illustrates the value of integrating evolutionary analyses of sequence variation with mechanistic appraisals of protein function. Investigation into the functional significance of the deer mouse ␤-globin polymorphism was motivated by the results of population genetic analyses which revealed evidence for a history of divergent selection between elevational zones. The experimental measures of O 2-binding properties corroborated the tests of selection by demonstrating a functional difference between the products of alternative alleles.A daptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. The tetrameric hemoglobin (Hb) protein, which transports O 2 from the respiratory surfaces to metabolizing tissues, is an ideal molecule for addressing questions about the functional evolution of allosteric, multimeric proteins. In jawed vertebrates, Hb is a heterotetramer, consisting of two ␣-chain subunits and two ␤-chain subunits that form two semirigid ␣␤ dimers (␣ 1 ␤ 1 and ␣ 2 ␤ 2 ). A mutual rotation of the two ␣␤ dimers occurs during the oxygenation-linked transition in quaternary structure between the deoxy-and oxyHb conformations that is basic to cooperativity (1). Because modifications of Hb function are often implicated in adaptation to environmental hypoxia, and because much is known about structure-function relationships of vertebrate Hbs and their role in blood-O 2 transport (2, 3), the study of Hb function in vertebrate species that are native to hypoxic environments provides an opportunity to elucidate detailed molecular mechanisms of p...

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Epistatic interactions between mutant sites in the same protein can exert a strong influence on pathways of molecular evolution. We performed protein engineering experiments that revealed pervasive epistasis among segregating amino acid variants that contribute to adaptive functional variation in deer mouse hemoglobin (Hb). Amino acid mutations increased or decreased Hb-O2 affinity depending on the allelic state of other sites. Structural analysis revealed that epistasis for Hb-O2 affinity and allosteric regulatory control is attributable to indirect interactions between structurally remote sites. The prevalence of sign epistasis for fitness-related biochemical phenotypes has important implications for the evolutionary dynamics of protein polymorphism in natural populations.

A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.