Darwin’s molecule

Chapter 9 discusses conceptual issues in protein evolution and provides a synthesis of lessons learned from studies of hemoglobin function. Using hemoglobin as a model molecule, we can exploit an unparalleled base of knowledge about structure-function relationships and we can characterize biophysical mechanisms of molecular adaptation at atomic resolution. It is therefore possible to document causal connections between genotype and biochemical phenotype at an unsurpassed level of rigor and detail. Moreover, since the oxygenation properties of hemoglobin provide a direct link between ambient O2 availability and aerobic metabolism, genetically based changes in protein function can be related to ecologically relevant aspects of organismal physiology. We therefore have a solid theoretical framework for making predictions and for interpreting observed associations between biochemical phenotype and fitness-related measures of whole-animal physiological performance. The chapter explores case studies that illustrate how experimental research on functional properties of a well-chosen model protein can be used to address some of the most conceptually expansive questions in evolutionary biology: Is genetic adaptation predictable? Why does evolution follow some pathways rather than others?

Evolution of the vertebrate globin gene family

Chapter 5 provides an overview of the evolutionary history of the globin gene superfamily and places the evolution of vertebrate-specific globins in phylogenetic context. The duplication and functional divergence of globin genes has promoted key physiological innovations in respiratory gas transport and other physiological functions during animal evolution. A combination of both tandem gene duplication and whole-genome duplication contributed to the diversification of vertebrate globins. Phylogenetic reconstructions arrange vertebrate globins into those that derive from vertebrate-specific duplications (cytoglobin, globin E, globin Y, and the independently derived myoglobin-like and hemoglobin-like genes of jawed vertebrates and jawless fishes [lampreys and hagfish]) and those that derive from far more ancient duplication events that predate the divergence between deuterostomes and protostomes (androglobin, globin X, and neuroglobin). Tracing the evolutionary history of deuterostome globins reveals evidence for the repeated culling of ancestral diversity, followed by lineage-specific diversification of surviving gene lineages via repeated rounds of duplication and divergence.

Allosteric theory

Chapter 3 provides a brief overview of allostery, the modulation of protein activity that is caused by an indirect interaction between structurally remote binding sites. In this mode of intramolecular regulatory control, the binding of ligand at a protein’s active site is influenced by the binding of another ligand at a different site in the same protein. This interaction at a distance is mediated by a ligation-induced transition between alternative conformational states. Hemoglobin is regarded as the “allosteric paradigm,” and the oxygenation-linked transition between alternative quaternary conformations provides a textbook example of how allostery works. This chapter reviews different theoretical models, such as the Monod-Wyman-Changeux “two-state” model, to explain the allosteric regulation of hemoglobin function.

Gene duplication and hemoglobin isoform differentiation

Chapter 6 explores the physiological significance of gene duplication and hemoglobin isoform differentiation. Repeated rounds of gene duplication and divergence during the evolution of jawed vertebrates promoted the diversification of the subfamilies of genes that encode the different subunit chains of tetrameric hemoglobin, leading to functional differentiation between hemoglobin isoforms that are expressed during different stages of prenatal development and postnatal life. The differentiation in oxygenation properties among developmentally regulated hemoglobin isoforms has clear adaptive significance in viviparous and oviviparous vertebrates alike. In some cases, a physiological division of labor between coexpressed isoforms may also contribute to the adaptive enhancement of tissue oxygen delivery.

Hemoglobin structure and allosteric mechanism

Chapter 4 provides an overview of hemoglobin structure and explains the mechanistic basis of ligand-binding dynamics and allosteric effects. The chapter provides an overview of decades of research on structure-function relationships based on X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. This body of work has provided atomic level insights into the structural mechanisms underlying key functional properties. The chapter also reviews experimental data on the ligand-binding behavior of hemoglobin, and discusses how the oxygenation properties of blood reflect a complex and dynamic interplay between the intrinsic O2-binding properties of hemoglobin and its operating conditions in the chemical milieu of the red blood cell.

The evolution of novel hemoglobin functions and physiological innovation

Chapter 7 explores the evolution of novel hemoglobin functions and physiological innovations. In the epic sweep of life’s history on Earth, globin proteins such as vertebrate hemoglobin were only recently co-opted for a respiratory function in circulatory O2 transport. Even after blood-O2 transport became an entrenched feature of vertebrate physiology, red blood cell hemoglobins evolved additional specializations of function in particular lineages. In some cases, like the Root effect of fish hemoglobins, these new functions represent key physiological innovations that have contributed to adaptive radiation. This chapter explores several case studies of how the evolution of novel allosteric properties have enhanced and expanded the physiological capacities of particular vertebrate groups, with an emphasis on teleost fishes and crocodilians.

A study in scarlet

Chapter 2 provides an overview of hemoglobin function and its physiological role in respiratory gas transport. Much of the chapter is devoted to explaining the physiological significance of cooperative oxygen binding by hemoglobin, and why it constitutes a key innovation in vertebrate evolution. The chapter explores the respiratory functions of hemoglobin under in vivo conditions in the red blood cell and its recently discovered role in regulating local blood flow via oxygen-coupled transport of vasoactive nitric oxide. Findings to date demonstrate that vertebrate hemoglobin has evolved physiologically important interactions with nitric oxide in addition to the more familiar interactions with oxygen and carbon dioxide.

Biochemical adaptation to environmental hypoxia

Chapter 8 explores mechanisms of hemoglobin adaptation to environmental hypoxia. Vertebrates living in hypoxic environments face the physiological challenge of optimizing the trade-off between oxygen loading at the respiratory surfaces and oxygen unloading in the tissue capillaries. In air-breathing and water-breathing vertebrates alike, fine-tuned adjustments in hemoglobin-oxygen affinity provide an energetically efficient means of compensating for a reduced oxygen tension of arterial blood. The adaptive significance of such changes is indicated by evolved changes in hemoglobin function in high-altitude mammals and birds, and erythrocytic acclimatization responses to environmental hypoxia in teleost fishes. An important goal for future research is to elucidate the specific physiological mechanisms by which changes in the oxygenation properties of hemoglobin translate into enhancements of whole-animal aerobic performance.

Principles of protein structure

Chapter 1 reviews basic principles of protein structure—the nature of proteins as polymers of amino acids, the variety of amino acids, and the way in which the physicochemical properties of amino acid side chains influence the folding of a polymer into a three-dimensional protein with specific functional properties. Whereas the main chain polypeptide is linked together by covalent bonds, the three-dimensional structure of native state proteins is mainly stabilized by a multitude of noncovalent, weakly polar interactions. After securing the base camp with this brief overview of protein structure, the subsequent chapters explore the functional properties of hemoglobin, the biophysical mechanisms underlying such properties, and the physiological role of hemoglobin in respiratory gas transport.