Elsevier

Fish Physiology

Volume 17, 1998, Pages 1-40
Fish Physiology

1 - Hemoglobin Structure and Function

https://doi.org/10.1016/S1546-5098(08)60257-5Get rights and content

Introduction

Transport of oxygen from the environment to cells and transport of metabolically produced carbon dioxide and H+ in the opposite direction are essential for vertebrate life. Hemoglobin (Hb) greatly increases the carrying capacity of O2, CO2, and H+ in blood as result of reversible binding of the ligands to the Hb molecule and appropriate allosteric interactions between the binding sites. With the notable exception of Antarctic icefishes, Hb is present in all vertebrates, which strongly decreases the circulatory requirement (i.e., volume of blood pumped by the heart for a unit quantity of O2 consumed).

Hemoglobin is one of the most intensively studied proteins, which has resulted in a deep understanding of its structure–function relationships. Hb has been termed the “honorary enzyme”, since the detailed knowledge of its structure and functions has rendered it a valuable model for studying allosteric interactions in other proteins. Research on vertebrate Hbs, and on fish Hbs in particular, has continued to reveal exciting new aspects of molecular and cellular control mechanisms. Significant advances have been made since fish Hbs were last reviewed in this book series (Riggs, 1970). The present chapter is intended to give an overview of the current knowledge of molecular structure, conformational changes, allosteric interactions, and the multiple functions of Hb in fish.

Section snippets

Basic Structure of Vertebrate Hemoglobins

Hemoglobin of most vertebrates is a tetrameric globular protein consisting of two α and two β polypeptide chains, each having an oxygen-binding heme (iron protoporphyrin IX). The number of amino acids varies slightly among species. In human Hb, the α chain has 141 amino acids, and the β chain has 146 amino acids. In carp, the numbers are 142 and 147, respectively. The amino acid sequences (primary structures) of a considerable number of Hbs are known, and they reveal homology that reflects

Ligand Binding to the Heme Groups and Its Allosteric Regulation

Reversible binding of oxygen to the heme groups demands that heme iron remains in the ferrous state and that binding of oxygen is favored over that of other potential heme ligands. Free heme groups in solution bind carbon monoxide 103–104 times as strongly as O2, whereas heme groups in myoglobin and hemoglobin bind CO only some 200 times more tightly than O2. The distal histidine is involved in this discrimination against CO in Mb and Hb, by sterically hindering bound CO and stabilizing bound O2

The Bohr Groups

The C-terminal His of the β chain (His HC3β) is the most important residue implicated in the pH regulation of Hbs. In human Hb, it is responsible for the Bohr effect observed in the absence of chloride ions and for about 40% of that measured in chloride-containing buffers (Shih et al., 1984, 1993). In the deoxygenated state, the carboxy group of His HC3β makes a salt bridge with the side chain of Lys C5α, while its imidazole ring is salt-bridged to the side chain of Asp FG1β (substituted by a

Hydrogen Ion Equilibria

The hydrogen ion equilibria of Hb are fundamental to both the structure and the physiological function of the protein. The exchange of H+ between protein and solvent is important for blood CO2 transport (by ensuring the necessary binding/release of H+ for the red cell CO2 hydration–dehydration reaction), and it makes Hb an effective nonbicarbonate buffer that limits fluctuations in blood pH upon acid or base additions. The H+ binding properties also determine the grouping of charges on the

Binding of Co2

CO2 reacts with uncharged α-amino groups to form carbamic acid. Since α-amino groups may be charged at physiological pH and since carbamic acid dissociates to carbamate, the following equilibria are involved: Hb—NH3+ ↔ Hb—NH2 + H+, Hb—NH2 + CO2 ↔ Hb—NHCOOH, Hb—NHCOOH ↔ Hb—NHCOO+ H+.

Carbamate formation is more pronounced in deoxygenated than in oxygenated Hb and is of physiological significance in mammals. In humans, oxygenation-dependent CO2 binding to Hb

Binding of Organic Phosphates

In anucleated mammalian red cells, the major organic phosphate influencing Hb function is 2,3-DPG, which is produced in anaerobic glycolysis. Nucleated fish red cells, in contrast, contain mitochondria and have an aerobic metabolism producing the nucleoside triphosphates (NTPs) ATP and GTP as potent allosteric effectors of Hb function.

Organic phosphates bind at the entrance to the central cavity between the two β chains in the T structure. Two amino acid substitutions change the site from one

Molecular Basis for the Root Effect

The Root effect (reviewed by Brittain, 1987) is characteristic of many (anodic) fish Hbs and is not found in other vertebrate Hbs. At low pH, Root effect Hbs show a large decrease in both oxygen affinity (i.e., a large Bohr effect) and cooperativity. The pH effect is so drastic that complete saturation with oxygen cannot be achieved even at very high oxygen pressures (140 atm; Scholander and Van Dam, 1954). Acidification of blood in the circulatory system will accordingly release large amounts

Temperature Effect

Oxygenation of Hb is exothermic, whereby Hb can be considered a heat carrier; the heat absorbed upon oxygen unloading in the tissues is liberated in the gills upon oxygenation. The apparent heat (or enthalpy) of oxygenation ΔHapp is usually calculated from the slope of log P50 versus 1/T plots,

ΔHapp=2.303RΔlogP50Δ(1/T),where R is the gas constant and T the absolute temperature (Wyman, 1964). ΔHapp includes the exothermic intrinsic heat of heme oxygenation but also contributions from heats of

Adaptation of Hemoglobin Function

The intrinsic (genetically coded) functional properties of hemoglobins determined by globin structure (amino acid sequence) result in species differences that may reflect adaptations to particular environments or metabolic requirements. This is exemplified by the high intrinsic Hb–O2 affinity in hypoxic-tolerant fish species and low intrinsic O2 affinity in active fish living in well-aerated water. Within a given species, the ability to transport O2, CO2, and H+ in the circulating blood can be

Hemoglobin Multiplicity

In contrast to humans (and most mammals) that have a single main Hb component, fishes commonly exhibit Hb multiplicity (different “isoHbs” that occur in the same individual at the same or different stages of its development) and Hb polymorphism (different “alloHbs” in genetically different strains of the same species). Hb multiplicity may result from either gene-related heterogeneity (variation in gene activity) or nongenetic heterogeneity (chemical modification in vivo or in vitro) (Kitchen,

Autoxidation

Autoxidation is the spontaneous oxidation of Hb (heme iron in oxidation state II) to metHb (heme iron in oxidation state III) by molecular oxygen. MetHb cannot bind O2 and accordingly is functionally inert with respect to oxygen transport. Free heme groups are rapidly and irreversible oxidized by O2 but the embedding of the heme groups in the globin effectively protects against autoxidation. Oxidation is promoted by anions such as CN or N3, which are strong ligands for ferric iron, thus

Interactions of Hemoglobin with Membrane Proteins

In mammalian erythrocytes, Hb binds to membrane components, and the interaction is mainly electrostatic. The most abundant membrane protein is band 3, which is present in about 1 million copies per RBC. Band 3 consists of a membrane domain, which mediates the physiological important anion exchange across the membrane, and a cytoplasmic domain, which is anchored to the cytoskeleton and to which hemoglobin and glycolytic enzymes also bind (Salhany, 1990). Binding of Hb occurs at the N-terminal

Acknowledgments

The authors acknowledge support from the Danish Natural Science Research Council (Centre for Respiratory Adaptation).

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