The 2.8 A crystal structure of hydroxylamine oxidoreductase of a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea, is described. Twenty-four haems lie in the centre bottom of the trimeric molecule, localized in four clusters within each monomer. The haem clusters within the trimer are aligned to form a ring that has inlet and outlet sites. The inlet is occupied by a novel haem, P460, and there are two possible outlet sites per monomer formed by paired haems lying within a cavity or cleft on the protein surface. The structure suggests pathways by which electron transfer may occur through the precisely arranged haems and provides a framework for the interpretation of previous and future biochemical and genetic observations.

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...

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.

Elucidating genetic mechanisms of adaptation is a goal of central importance in evolutionary biology, yet few empirical studies have succeeded in documenting causal links between molecular variation and organismal fitness in natural populations. Here we report a population genetic analysis of a two-locus α-globin polymorphism that underlies physiological adaptation to high-altitude hypoxia in natural populations of deer mice, Peromyscus maniculatus. This system provides a rare opportunity to examine the molecular underpinnings of fitness-related variation in protein function that can be related to a well-defined selection pressure. We surveyed DNA sequence variation in the duplicated α-globin genes of P. maniculatus from high- and low-altitude localities (i) to identify the specific mutations that may be responsible for the divergent fine-tuning of hemoglobin function and (ii) to test whether the genes exhibit the expected signature of diversifying selection between populations that inhabit different elevational zones. Results demonstrate that functionally distinct protein alleles are maintained as a long-term balanced polymorphism and that adaptive modifications of hemoglobin function are produced by the independent or joint effects of five amino acid mutations that modulate oxygen-binding affinity.

SUMMARY In high-altitude vertebrates, adaptive changes in blood–O2 affinity may be mediated by modifications of hemoglobin (Hb) structure that affect intrinsic O2 affinity and/or responsiveness to allosteric effectors that modulate Hb–O2 affinity. This mode of genotypic specialization is considered typical of mammalian species that are high-altitude natives. Here we investigated genetically based differences in Hb–O2 affinity between highland and lowland populations of the deer mouse (Peromyscus maniculatus), a generalist species that has the broadest altitudinal distribution of any North American mammal. The results of a combined genetic and proteomic analysis revealed that deer mice harbor a high level of Hb isoform diversity that is attributable to allelic polymorphism at two tandemly duplicated α-globin genes and two tandemly duplicated β-globin genes. This high level of isoHb diversity translates into a correspondingly high level of interindividual variation in Hb functional properties. O2 equilibrium experiments revealed that the Hbs of highland mice exhibit slightly higher intrinsic O2 affinities and significantly lower Cl– sensitivities relative to the Hbs of lowland mice. The experiments also revealed distinct biochemical properties of deer mouse Hb related to the anion-dependent allosteric regulation of O2 affinity. In conjunction with previous findings, our results demonstrate that modifications of Hb structure that alter allosteric anion sensitivity play an important role in the adaptive fine-tuning of blood–O2 affinity.

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.

The carbonic anhydrases (CAs) fall into three evolutionarily distinct families designated ␣-, ␤-, and ␥-CAs based on their primary structure. ␤-CAs are present in higher plants, algae, and prokaryotes, and are involved in inorganic carbon utilization. Here, we describe the novel x-ray structure of ␤-CA from the red alga, Porphyridium purpureum, at 2.2-Å resolution using intrinsic zinc multiwavelength anomalous diffraction phasing. The CA monomer is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an ␣/␤ domain and three projecting ␣-helices. The motif is obviously distinct from that of either ␣-or ␥-CAs. This homodimeric CA appears like a tetramer with a pseudo 222 symmetry. The active site zinc is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the ␤-CAs. No water molecule is found in a zinc-liganding radius, indicating that the zinc-hydroxide mechanism in ␣-CAs, and possibly in ␥-CAs, is not directly applicable to the case in ␤-CAs. Zinc coordination environments of the CAs provide an interesting example of the convergent evolution of distinct catalytic sites required for the same CO 2 hydration reaction.Carbonic anhydrase (CA, 1 EC 4.2.1.1) is a zinc-containing enzyme which catalyzes the reversible hydration of CO 2 . CA is ubiquitously distributed in nature and is involved in fundamental biological processes such as photosynthesis, respiration, pH homeostasis, and ion transport (1, 2). Based on sequence homologies, CAs are classified into three evolutionarily distinct groups, designated ␣-, ␤-, and ␥-CAs (3). ␣-CAs are found in mammals, algae, and prokaryotes, ␤-CAs in higher plants, algae, and prokaryotes, and ␥-CA has been identified in the archaeon, Methanosarcina thermophila, although gene homologies have been recently found in higher plants and prokaryotes (4).The x-ray structures of ␣-CAs from several mammals (5, 6) have revealed a common overall structure dominated by ␤-sheets. The catalytically active zinc is liganded by three histidine residues with a hydroxide or a water molecule as a fourth ligand, giving a tetrahedral coordination geometry. For the CO 2 hydration reaction, the zinc-bound hydroxide initiates a nucleophilic attack on the substrate CO 2 to form zinc-bound HCO 3 Ϫ which is then displaced by a water molecule (7). The regeneration of zinc-bound hydroxide requires an intramolecular proton shuttle from the zinc-bound water to the bulk solvent for each cycle of catalysis. The proton transfer is ratelimiting in the catalysis (8).The x-ray structure of ␥-CA from M. thermophila has been reported (4). This is a trimeric CA with a peculiar left-handed ␤-helical folding motif. The active site zinc is located at subunit interfaces and coordinated by two histidines from one subunit and one histidine from a neighboring one. In addition, at an electron dense region which occupies the fourth coordination site, a putative water molecule is recognized. Although the polypeptide assembly forming the active site of the ...

Animals that sustain high levels of aerobic activity under hypoxic conditions (e.g., birds that fly at high altitude) face the physiological challenge of jointly optimizing blood-O 2 affinity for O 2 loading in the pulmonary circulation and O 2 unloading in the systemic circulation. At high altitude, this challenge is especially acute for small endotherms like hummingbirds that have exceedingly high mass-specific metabolic rates. Here we report an experimental analysis of hemoglobin (Hb) function in South American hummingbirds that revealed a positive correlation between Hb-O 2 affinity and native elevation. Protein engineering experiments and ancestral-state reconstructions revealed that this correlation is attributable to derived increases in Hb-O 2 affinity in highland lineages, as well as derived reductions in Hb-O 2 affinity in lowland lineages. Site-directed mutagenesis experiments demonstrated that repeated evolutionary transitions in biochemical phenotype are mainly attributable to repeated amino acid replacements at two epistatically interacting sites that alter the allosteric regulation of Hb-O 2 affinity. These results demonstrate that repeated changes in biochemical phenotype involve parallelism at the molecular level, and that mutations with indirect, second-order effects on Hb allostery play key roles in biochemical adaptation.high-altitude adaptation | hypoxia | parallel evolution | protein evolution | epistasis