Common Biochemical Pathway

As might be expected from their common structure, all cellular tetrapyrroles are

Figure 2. The structure of protoheme IX.

Hemes are found in a wide range of different proteins, including photosynthetic and respiratory cytochromes involved in electron transfer, the oxidative enzymes catalase and peroxidase, cytochrome P450s, which catalyze mono-oxygenase reactions, and oxygen-carrying proteins such as hemoglobin and myoglobin (Chapters 7, 8, and 9).

Figure 2. The structure of protoheme IX.

Hemes are found in a wide range of different proteins, including photosynthetic and respiratory cytochromes involved in electron transfer, the oxidative enzymes catalase and peroxidase, cytochrome P450s, which catalyze mono-oxygenase reactions, and oxygen-carrying proteins such as hemoglobin and myoglobin (Chapters 7, 8, and 9).

made by a common biochemical pathway (Figure 4) from the central intermediate uroporphyrinogen III (for a review see Reference 37).

The first committed precursor is ALA, which contains all of the carbon and nitrogen atoms required by the tetrapyrrole nucleus. Two biosynthetic pathways that lead to ALA have evolved (Chapter 4). The first pathway to be discovered was the so-called Shemin pathway, in which ALA is formed from glycine and succinyl CoA by ALA synthase (ALAS). This occurs in animals, fungi, and some bacteria. However, the ancestral pathway, characteristic of the majority of bacteria, algae, and plants, is the C5 pathway, in which ALA is formed from glutamate in three steps involving glutamyl-tRNA as an intermediate. Monomers are formed by condensation of ALA by ALA dehydratase (Figure 5) to form porphobilinogen (PBG), which is in turn tetramerized by PBG deaminase (Figure 6) to form the linear intermediate 1-hydrox-ymethylbilane (or preuroporphyrinogen). This is cyclized and isomerized by uropor-phyrinogen III synthase, to produce the common intermediate to all cellular tetrapyrroles.

Reduced uroporphyrinogen III is formed with methylene rather than meth-ine bridges to prevent photoactivity, production of singlet oxygen, and similar damaging species. The porphyrinogen form is maintained until the step preceding metal ion insertion. Uroporphyrinogen III has two possible fates. On the corrin pathway, it is methylated and used to produce siroheme, the cofactor of sulfite and nitrite reductases, or vitamin B12, after the insertion of ferrous iron or cobalt, respectively. Alternatively, uroporphyrinogen III is oxidatively decarboxylated in three steps to form protoporphyrin IX, the last common intermediate of heme and chlorophyll (Chapters 4 and 5).

Ferrochelatase catalyzes insertion of iron into protoporphyrin IX for heme biosynthesis, which is followed by insertion into protein complexes. Heme may be metabolized further to form bilins (Chapters 12-14) by linearization and the loss of the iron atom, catalyzed by heme oxygenase. The insertion of magnesium into protoporphyrin IX by magnesium chelatase is the first step of chlorophyll biosynthesis and is followed by further modification of the tetrapyrrole nucleus by esterification, methylation,

Figure 3. The structure of phytochromobilin. This is the chromophore of phytochromobilin, which is the red-light receptor of higher plants (Chapter 13). Linear tetrapyrroles are also found as accessory light-harvesting pigments in cyanobacteria and many algae (Chapter 14).
Figure 4. The tetrapyrrole biosynthetic pathway, showing the different endproducts and the major intermediates (Chapters 4, 5, 6, and 12). Enzymes are shown in italics.

reduction of vinyl group, and formation of a fifth ring to produce protochlorophyllide. In the presence of light, protochlorophyllide is reduced to form chlorophyllides, which undergo esterifiication by phytyl diphos-phate or geranylgeranyldiphosphate to produce chlorophyll (see Chapter 6).

3. ROLES OF TETRAPYRROLES 3.1. Light Harvesting

Photosynthetic organisms contain a sophisticated system of several hundred chlorophylls (or bacteriochlorophylls) and other accessory pigments, which act as antennae to absorb light and pass the energy to special chlorophylls in reaction centers. Here the light energy is trapped as excited electrons, which are then transferred through an electron transfer chain to generate ATP. In higher plants, algae, and cyanobacteria, this process results in the oxidation of water to evolve molecular oxygen and the production of reduced nicoti-namide adenine dinucleotide phosphate (NADPH) (see Chapters 10 and 11 for more detail). The ATP and NADPH generated by the light-dependent reactions are used to fix CO2 into organic combination via the Calvin cycle. Photosynthesis not only provides the means for photosynthet-ic organisms to live, but also indirectly supports almost all life on earth with carbohydrates and oxygen.

3.2. Oxidation of Carbohydrates to Produce Usable Energy

Nonphotosynthetic cells obtain their energy by the oxidation of carbohydrates, which in aerobic organisms results in the formation of CO2. This process involves a series of reduction-oxidation (redox) reactions whereby the large gap in oxidation state between carbohydrate and carbon dioxide is released in a series of gentle and efficient steps, with oxygen as the final electron acceptor. Transition metals, such as the iron found in heme (Figure 2), are well suited to catalyze these reactions because they contain ^-electron orbital systems with small differences in energy levels, thus allowing a range of oxidation states so that energy can be released in a controlled and useful way (cf section 3.4).

In the bacterial membrane, and the mitochondria of eukaryotes, a series of protein complexes containing cofactors, which include heme (see Chapter 7), undergo a series of reversible redox reactions that generates ATP. In this respect, the process of

Figure 5. The formation of a pyrrole. The reaction catalyzed by ALA dehydratase.
Figure 6. The formation of a tetrapyrrole. The reaction catalyzed by PBG deaminase. The holoenzyme (E) contains an active site dipyrromethane cofactor. This is used to accept PBG monomers and form enzyme substrate complexes. A, acetate; P, propionate moiety. Pyrrole rings are labeled A, B, C, and D.

oxidative phosphorylation is similar to that in photosynthesis (section 3.1), reflecting common evolutionary ancestors of some of the components involved.

3.3. Transport and Homeostasis of Oxygen

Metabolism requires the consumption of large amounts of oxygen, and therefore transport from the atmosphere to cells buried deep in animal bodies is necessary to allow a rapid rate of metabolism. While several kinds of oxygen transport proteins exist, including hemerythrins (non-heme iron proteins) and hemocyanins (copper proteins), globins are the most abundant and widespread oxygen transport protein (12). They use reversible oxygen coordination to protoheme IX iron (Fe11) for transport of oxygen from respiratory organs throughout the animal body.

Hemoglobins transport oxygen in many animal groups, and most have similar structures and functions (35). In circulating red blood cells, hemoglobins consist of 17 kDa polypeptides, each containing a heme group that can bind one oxygen molecule. Hemoglobins are typically tetramers that allow for cooperative binding and release of oxygen, depending on the Pq2 of the surrounding tissue.

In addition to oxygen transport, hemoglobins are associated with other important functions. For example, circulating hemoglobin I of the swamp clam Lucina pectina-ta is involved in sulfide transport. Sulfides are bound with high affinity within a cage of heme and three phenylalanine residues and are released to symbiotic bacteria upon reduction (3).

Myoglobin is a monomeric protein that contributes to the transport of oxygen by diffusion, and large amounts are found in skeletal and cardiac muscles of mammals. In cardiac muscle, myoglobin acts as a short-term oxygen buffer, smoothing sup ply from one beat to the next. Intracellular myoglobin is also found in bacteria, protozoans, plants, and invertebrates (32).

Plants also contain leghemoglobin used for the homeostasis of oxygen. The best studied example is in the legume root nodule, where symbiotic bacteria consume large amounts of energy during the reduction of atmospheric nitrogen to ammonia, which is then available to the host plant. Leghemoglobin facilitates the maintenance of high levels of oxygen needed for bacterial respiration, while preventing poisoning of the nitrogen fixing machinery by molecular oxygen (2). Unlike in animals, the protein is not circulated, but rather simple diffusion down an oxygen gradient created by bacterial respiration promotes transport of oxygen from the outside.

3.4. Protection of Cellular Processes from Reactive Oxygen Species

As well as the desired biological reaction of oxygen as a terminal acceptor of electrons in the controlled oxidation of carbohydrates in respiration, oxygen will also react with electrons encountered at random, to produce reactive oxygen species (ROS) such as oxygen radicals and superoxide. These are very harmful to living systems, causing lipid peroxidation, membrane damage, and genetic mutation. Cells contain a number of enzymes that act to remove these intermediates quickly. Many of these oxidases are heme-containing proteins, including peroxidases and catalase (7).

3.5. Taking Advantage of Reactive Oxygen Species

Although ROS can be harmful, organisms have evolved systems in which they are useful. One important example of this is the biosynthesis and degradation of lignin. Lignocellulose is a composite of lignin and cellulose and probably the most abundant organic molecule in the biosphere, functioning as material for mechanical strength in wood. Rather than a regular polymer such as cellulose, lignin is synthesized by peroxidases, which oxidize phenol-propane units (such as coniferyl alcohol) to form reactive radical species. These polymerize in an irregular fashion to form lignin, which thereby contains a wide array of chemical linkages (38). Because of this unsymmetrical arrangement, lignin is unusually resistant to enzymatic breakdown, and only a few microbes, such as the white rot fungi, have enzymes capable of doing so (20). Lignin is also an important byproduct of the paper industry, which generates about 30 million tons of unused lignin per year, in a process involving harsh chemical treatments (13). Genetic modification of tree species to reduce lignin content is being explored as a means of avoiding this costly and polluting process (34).

A more general, and essential, role of these reactive species is found in both plants and animals, where ROS have been shown to modulate a wide range of cellular and physiological processes, acting as part of the signal transduction pathway. For example, one of the earliest responses to pathogen attack is a marked increase in ROS in the infected tissue, produced in part by the activity of plasma membrane-bound NADPH oxidase, a hemoprotein complex. The ROS, in turn, act as a trigger for defense responses, such as modification of membrane permeability and ion fluxes, and systemic acquired resistance in plants (1). Similarly in animals, ROS influence signaling cascades and transcriptional— posttranscriptional control of gene expression, thereby playing an essential role in processes such as apoptosis (23).

3.6. Control of Metabolic and Cellular Processes by Signaling

As well as functioning as an enzymic prosthetic group, tetrapyrroles also function in key regulatory processes. These include control of gene expression, cellular signaling pathways, and control of protein transport within the cell.

An example is heme-mediated feedback control of its own synthesis, which appears to occur in all groups of organisms, most importantly at the production of the first committed precursor ALA. In plants, there is evidence that heme inhibits the enzyme glutamyl-tRNA reductase (36). In animals, although heme inhibits the activity of ALAS, control is exerted at several other points as well. In mammals, there are two forms of the enzyme, constitutive and ery-throid-specific. Liver ALAS is inhibited by heme in a negative feedback loop (11) to maintain levels of heme production for the maintenance of cellular processes. This feedback regulation is achieved by a combination of effects including inhibition of ALAS gene expression, increased ALAS mRNA degradation, and inhibition of pre-ALAS protein transport to the mitochondrion (19), with only a minor contribution by inhibition of ALAS catalytic activity (28). In contrast to the liver, in erythroid cells, transcription of the ALAS gene, together with genes for later enzymes in the pathway and for globins, is stimulated by heme to produce the large amounts of heme needed for hemoglobin in red blood cells.

In yeast, expression of the ALAS gene is controlled by heme and mediated through the transcription factor HAP1, which binds heme for activity. The binding domain contains multiple copies of a short motif, which is also found in the mito-chondrial transit peptide of mammalian ALAS. This motif, involved in transient binding of heme, is quite different to the more stable heme-binding domain of cytochromes and globins (39).

There is accumulating evidence that tetrapyrrole intermediates play a role in signaling. In plants, there is coordination between the chloroplast and the nucleus, such that nuclear-encoded genes for chlo-roplast-targeted proteins are transcribed only in cells with functional chloroplasts. Although the exact identity of the so-called "plastid-factor" remains elusive, plant mutants with defects in certain steps of the tetrapyrrole biosynthetic pathway have altered plastid-nuclear signaling (24).

3.7. Subtle Pigmentation

While plants are green because of the presence of chlorophyll and animal tissues are largely red due to heme, some of the more subtle animal colors are also conferred by tetrapyrroles. The cuticle of birds' eggs with colored shells contain tetrapyrroles which contribute to their camouflage. Most markings and pigmentation are due to pro-toporphyrin IX, which is associated with brown and black coloring. Blue eggshells are associated with biliverdin IXa, and green eggshells are associated with zinc-biliverdin IXa with traces of copropor-phryin III (18). The feathers of some birds also contain tetrapyrrole pigments. The feathers of Turocos contain red turacin (cop-per-uroporphyrin III) and green tura-coverdin (22). Uroporphryin I is found in many calcified mollusk tissues such as shells (17) and pearls (16), though the function of the tetrapyrrole is unknown.

3.8. Artificial Uses of Tetrapyrroles

In addition to their importance in biology, tetrapyrroles are increasingly of interest to a much wider range of researchers. For example, chemists are able to create synthetic molecules which mimic the recognition and catalytic properties of enzymes. A particular aspect of this work is catalysis of reactions for which there are no known natural enzymes, such as Diels-Alder reactions (4). Porphyrins have proved very useful for this sort of study because of the rigid structures that they are able to form and the fact that they can coordinate a number of metal ions which are involved in the catalysis. For example, using porphyrin molecular boxes and zinc coordination, it has been possible to influence the stereospecificity of reactions by the geometrical constraints of a host cavity (26).

Other novel uses of tetrapyrroles have been established in clinical medicine, in particular for the treatment of cancer cells, in a technique called photodynamic therapy (PDT). The rationale behind the method is to load the cancerous cells with photosensitizing porphyrin mixtures, which, upon irradiation with visible light, cause the production of singlet oxygen, thereby leading to the destruction of the cells as described in section 3.4. Porphyrins are ideal compounds for this technique, not only because of their light absorption properties, but also because there is some preferential uptake of these molecules by tumor cells. Initially, in the 1960s and 1970s, the major photosensitizers used were hematoporphyrins and related preparations derived from acid extraction of blood (or hemoglobin), and therefore, are not chemically defined compounds. However, since 1980, new sensitizers have been developed, including chlorins and phthalo-cyanines, which have been chemically synthesized (5).

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