American Scientist

One Ring to rule them all,
One Ring to find them,
One Ring to bring them all
and in the darkness bind them.

—J. R. R. Tolkien, The Fellowship of the Ring

The porphyrin pathway is ubiquitous in the biological realm, serving throughout the plant and animal kingdoms as the assembly line for the most abundant pigments in nature. The ring-shaped porphyrin molecules bind an array of metal ions, with each combination associated with different biological functions. Chlorophylls bind magnesium to play a pivotal role in photosynthesis. Heme binds iron to coordinate molecular oxygen and carbon-dioxide transport, supports the electron-transport chains necessary for cellular respiration and contributes to the catalytic activities of many enzymes. Porphyrins bind nickel to form coenzyme F₄₃₀, which plays critically important roles in bacteria that metabolize methane. Vitamin B₁₂ is formed from the binding of cobalt to a derivative of porphyrin; lack of the vitamin can result in pernicious anemia and impair the function of the brain and nervous system. Taken together, these porphyrin-derived pigments can be called the “colors of life,” in the sense that these rings are necessary to sustain key activities in nearly all organisms.

It should come as no surprise then, that when the porphyrin pathway is disrupted, there can be remarkably far-reaching consequences. Derangements of porphyrin metabolism may have left their marks on the legacy of King George III and the founding of the United States, and on the legend of Dracula. Disruption of porphyrin metabolism can also be useful—for example, in the development of herbicides.

The Key Enzyme

The early porphyrin precursors differ between plants and animals, but they converge at the first committed molecule in the chain of synthesis, delta-aminolevulinic acid. And the subsequent steps involved in the synthesis pathway are universal (see Figure 2). Protoporphyrinogen oxidase is the last enzyme before a major branching point in the synthesis of the “rings of life.” The next step finds enzymes catalyzing the binding of various metal ions to protoporphyrin, committing the master ring to the synthesis of an array of biologically important molecules.

In animal cells, protoporphyrinogen oxidase is bound to the inner membrane of mitochondria, the cell’s powerhouses. Plants possess two evolutionarily distinct genes, encoded in the nucleus, that create two forms of the enzyme, one found on the outer membrane of chloroplasts (the light-gathering organelles of plant cells) and the other again located in mitochondria. These enzymes have only 25 percent of their amino acids in common and are flanked by different transit peptides, which are short stretches of amino acids that direct newly synthesized proteins to specific locations in the cell.

Molecules of a plant’s mitochondrial protoporphyrinogen oxidase are loosely associated (see Figure 3). Each molecule, or monomer, possesses three domains: One binds the substrate, the second the membrane and the third an oxidation-reduction factor important in metabolism called flavin adenine dinucleotide (FAD). FAD can accept electrons from upstream metabolites and pass them downstream, making it a key helper in the production of energy in the cell. FAD acts like a roof on the active site. Within the active-site domain, a glycine probably participates in positioning the target substrate, protoporphyrinogen. Some have proposed that another amino acid, arginine, participates in ionic interactions with one of the side chains of protoporphyrinogen, and it may coordinate the orientation of the substrate with respect to the FAD cofactor.

Protoporphyrinogen oxidase catalyzes the removal (or oxidation) of six hydrogen atoms from the colorless substrate to form the photodynamic red pigment protoporphyrin IX. The reaction is not well understood, but it involves three sequential FAD-driven transfers of hydrogen at the central ring of protoporphyrinogen, followed by a complex hydrogen rearrangement. The position of the glycine relative to the opposing FAD ring is critical to the rate of catalysis, as it controls the distance between the carbon ring of the substrate and the FAD cofactor.

The details of the workings of this enzyme pathway are vital to understanding disorders in the production of different porphyrins. Problems with production of heme are particularly significant to humans.

In the human body, heme is synthesized primarily in the liver and bone marrow, and most of it is destined to become bound in the hemoglobin of red blood cells. Accumulation of the purple-red porphyrin intermediates outside of their normal locations is symptomatic of a group of diseases collectively referred to as porphyria. In Greek, porphuros means purple. There are genetically inherited dysfunctions associated with each of the steps of heme synthesis shown in Figure 2. Cutaneous porphyrias cause light-dependent swelling and itching of the skin that may develop into rashes and blisters. Acute porphyrias affect the nervous system, in part via deficiency in porphyrin-derived vitamin B₁₂, inducing mental disorders ranging from subtle dysphoria to severe disturbances, as well as engendering pain, muscle numbness and vomiting. Acute porphyria is a term that includes three similar genetically inherited diseases: acute intermittent porphyria, hereditary coproporphyria and variegate porphyria. The development of acute symptoms can be triggered by exposures to foreign substances such as heavy metals, polyhalogenated aromatic hydrocarbons (a type of environmental pollutant) and other compounds that interfere with the porphyrin pathway.

Variegate porphyria is linked to a dysfunction of protoporphyrinogen oxidase. People with this disorder inherit one defective allele, resulting in approximately half the normal activity of protoporphyrinogen oxidase. Patients suffering from variegate porphyria may exhibit both neuropsychiatric symptoms and skin lesions associated with chronic photodermatitis, in addition to increased levels of porphyrins in the feces. Approximately three-quarters of those with the defective allele remain asymptomatic and lead normal healthy lives, but these individuals remain at risk for episodes of illness and must take simple measures to avoid certain triggering factors. Even the few who do become ill usually make a complete recovery and have fewer attacks as they age. However, acute attacks can be very severe in some individuals, and the disorder may affect lives far beyond those who inherit the disease.

The Madness of King George

The water is of a deeper colour—and leaves a pale blue ring upon the glass near the upper surface.

—Remarks of Sir Henry Halford, one of the attending physicians of King George III, on the monarch’s urine (January 6, 1811).

King George III, monarch of England from 1760 until his death in 1820, was one of the longest-serving British rulers. During his reign, the British Empire was well on its way to exerting economic dominance over international trade and military supremacy over the oceans. This rise in prominence was, however, tarnished by a strange sickness afflicting the English monarch, whose sometimes irrational decisions may have led to the American Revolutionary War and the subsequent loss of the American colonies.

Royal medical records are replete with well-documented episodes of an incapacitating sickness that plagued King George. In particular, five major episodes each included a prolonged illness in which physical abnormalities were accompanied by profound degradation of his mental faculties. The King’s irrational behavior was so debilitating during one episode, which lasted from October 1788 to February 1789, that it instigated a constitutional review known as the Regency Crisis.

These periods of mental incapacity were originally thought to result from a psychiatric illness, but a more precise diagnosis—that the King suffered from porphyria—has been proposed by a number of researchers since the mid-1960s. Indeed, medical records provide evidence that King George exhibited all of the symptoms typically associated with the disease, such as strong abdominal pain, port-wine–colored urine and paralysis in the arms and legs. Initially, it was suggested that the King suffered from acute intermittent porphyria. However, a follow-up study that examined the medical records of forebears, descendants and collateral relatives of King George III led to the diagnosis of variegate porphyria.

King George III may have suffered from a particularly severe form of porphyria. He experienced his first attack in 1765, four years after his marriage to Charlotte of Mecklenburg-Strelitz. Further signs of the disease were recorded by his physicians between 1788 and 1789. From 1811 until his death in 1820, the King’s health continued to decline. The disease eventually impaired his faculties to the extent that he required constant care and was confined to his private apartments at Windsor Castle.

Variegate porphyria is an inherited condition, but acute attacks may be triggered by exposure to toxic metals. If King George III did, indeed, carry the genetically defective protoporphyrinogen oxidase, he would have been hypersensitive to the effects of heavy metals and other environmental contaminants common in England during this period. Elemental analysis of the King’s hair detected normal to slightly elevated levels of mercury and lead, but levels of arsenic reflecting systemic toxicity were also measured in the King’s hair by Timothy J. Cox of the University of Cambridge and his colleagues in 2005. Therefore, arsenic poisoning may have triggered King George III‘s recurring severe porphyria episodes.

In 1968 British physician Ida MacAlpine and her colleagues studied the medical records of 13 generations of members of King George III’s lineage spanning more than 400 years. They found biochemical evidence that the King and numerous other members of the royal line may have suffered from porphyria (see Figure 4). Several other family members may have been carriers of the genetic defect, but their medical records do not suggest that they exhibited any symptoms of porphyria. This lack of evidence is not surprising because variegate porphyria is a low-penetrance disorder, and 75 percent of carriers are asymptomatic. Subsequent studies in 1982 by Lindsay C. Hurst of Moorhaven Hospital in England have suggested that porphyria can be traced back even further to Henry VI of England and Charles VI of France in the 14th and 15th centuries. A 1996 study by Martin J. Warren, of the University of Kent, and his colleagues looked at more recent generations and found a link to the disease in the descendants of King George III.

Porphyria’s grip on King George affected both his physical and mental health, which may have had a tremendous impact on the course of history. Following the French and Indian War, which ended in 1763, Britain levied a series of taxes and enacted several laws to strengthen its authority over its American colony. The colonists, who were British subjects, objected to this assertion of power and famously complained that there should be “no taxation without representation.” As political tensions heightened, the King took increasingly strident positions, such as sending British troops to squash the Boston tea party rebellion. He also removed local governments and installed royal officials in their stead. Patriots mobilized their militias, and the American Revolutionary War started in 1775. Historical accounts show that the King obstinately (perhaps even irrationally) resisted any diplomatic solutions that were pursued throughout the duration of the war. This outcome eventually led the colonists to declare independence from British rule in July 1776. The text of the Declaration of Independence itemizes King George’s unwillingness to compromise:

Such has been the patient sufferance of these Colonies; and such is now the necessity which constrains them to alter their former Systems of Government. The history of the present King of Great Britain is a history of repeated injuries and usurpations, all having in direct object the establishment of an absolute Tyranny over these States.

The war essentially ended with the surrender of Lord Cornwallis at Yorktown on October 19, 1781, followed by formal British abandonment of any claims to the United States with the Treaty of Paris in 1783.

Certain historians have postulated that porphyria paralyzed the energies of King George III, clouding his judgment and making him temperamental. Such views have also been popularized in the film The Madness of King George. Mood swings and impaired reason led to a number of unwise and rash decisions that would ultimately sever the ties to British subjects living in the American colonies, to the detriment of both Britain and loyalists in America. These historians conjecture that had King George III not been incapacitated by illness, he might have been able to avoid the American Revolutionary War.

It should be noted, however, that despite the medical records suggesting that the King suffered from recurrent attacks of porphyria, this retrospective diagnosis is still the subject of dispute. Two studies, published in 2010 by Timothy J. Peters at the University of Birmingham and his colleagues, reexamined the King’s medical records from clinical and psychiatric perspectives, and they concluded that he might have instead suffered manic-depressive psychosis. The source of the King’s malady may never be conclusively proven, but there are other interesting stories that may have been marked by porphyria.

Vlad Tepes and Dracula

Then, with the fear on me of what might be, I drew a ring so big for her comfort, round where Madam Mina sat. And over the ring I passed some of the wafer, and I broke it fine so that all was well guarded. She sat still all the time, so still as one dead.

—Bram Stoker, Dracula

Vlad III, born in 1431, was Prince of Wallachia (a region of Romania) during the mid- to late-15th century. A descendent of Mircea the Great, King of Wallachia from 1386 until 1418, Vlad was Knight of the Order of the Dragon, a religious and chivalric order created in Hungary whose purpose was to protect the interests of Catholicism and the Holy Roman Empire, especially against the Ottoman Empire. Vlad was an authoritarian ruler who built a strong military organization that permitted increased and safer trade with neighboring countries.

Vlad was extremely cruel to his foes, whether fellow countrymen or soldiers of invading armies. On Easter Sunday 1459, he arrested and impaled many of his nobles who had rebelled and killed his father and his brother. His preference for this form of punishment consequently earned him the nickname Tepes, which means “the impaler.” Vlad Tepes’s appetite for torturing his enemies seems to have been bottomless. He condemned people to death using a myriad of gruesome methods such as skinning, decapitation, hacking, strangulation, hanging, boiling and burning. Other punishments included cutting off noses, ears, sexual organs and limbs. Vlad Tepes is said to have killed 20,000 to 40,000 European civilians, mostly by impalation.

Having been captured and tortured by the Ottoman Empire during his childhood, Vlad was particularly ruthless toward invading Muslim armies. In 1462, when Sultan Mehmed the Conqueror pursued Vlad, he encountered a “Forest of the Impaled” as he entered the capital of Wallachia, where thousands of stakes held the rotting cadavers of 20,000 Turkish captives. In all, Vlad Tepes is said to have impaled as many as 100,000 Turkish Muslims.

Although his bloodthirstiness is not known to have extended to literal ingestion of his enemies’ blood, Vlad Tepes also served as the model for an enduring fictional villain, Dracula. Mythological “undead” beings who feed on the blood of the living have been recorded in most cultures from the earliest times. The term vampire, however, was not developed in Western Europe until Eastern European vampire legends were popularized during the early 18th century. Bram Stoker, an Irish writer, first heard of Vlad Tepes in 1890 during an encounter with the Hungarian professor Ármin Vámbéry. Stoker became fascinated by the prince’s perplexing personality and developed his phantasmagoric fictional character of Dracula the vampire from the vivid and gruesome historical accounts of Vlad’s cruelty. He worked on his novel for several years and published Dracula in May 1897. Stoker’s choice of the name Dracula is also extracted from the life of the prince of Wallachia. Vlad and his father were both Knights of the Order of the Dragon. In the Romanian language, “Dragon” is pronounced “Dracul,” and the noble families of Romania called Vlad’s father Dracul. Dracula simply means “the son of Dracul,” and Vlad ultimately adopted this as a nickname.

There is no evidence that Stoker knew about porphyria or the striking similarity between the appearances of the character he created and some of the symptoms commonly associated with the disease. (There is also no indication that Vlad Tepes suffered from porphyria.) However, David Dolphin, a Canadian chemist who edited an authoritative seven-volume series on porphyrins, realized the similarity between the imaginary character of Dracula and some of the symptoms of patients affected by porphyria.

Dolphin postulated that these stories may have been derived from the mythologization of actual individuals affected by porphyria. Indeed, the accumulation of photodynamic pigments in the epidermis and retina renders porphyria victims exceedingly sensitive to light. Such harmful effects can lead to disfigurations such as the withering of fingers and lips, and gums may tighten to reveal fanglike teeth with reddish hues due to elevated porphyrin levels.

Although it is possible that a dysfunction in protoporphyrinogen oxidase may prove to be a scientific basis that contributed to the creation of the myth of vampires, it is important to remember that patients afflicted with porphyria are by no means vampires and that it is certainly an unfortunate coincidence that this serious malady has been connected to such a dreadful, fictitious creature. No one suffering from porphyria deserves a rendezvous with Buffy the vampire slayer. Instead, its victims should be able to seek medical attention and not feel stigmatized.

Porphyrias in Plants

Thy design I know well,
But little I care:
Who wins the ring
Will rule by its might.

—Richard Wagner, The Ring of the Nibelung

Porphyrias in plants have largely gone unnoticed because dysfunction in the highly regulated porphyrin pathway probably carries a serious fitness penalty, and affected plants most likely do not survive. Gongshe Hu and colleagues at the University of Missouri have, however, recently characterized a maize line called Les22 exhibiting light-dependent lesions on its foliage. Les22 was cloned using a mutator-tagging technique, and analysis revealed that it encodes uroporphyrinogen decarboxylase, a key enzyme in the biosynthetic pathway of chlorophyll and heme. Mutations in uroporphyrinogen decarboxylase are associated with porphyria cutanea tarda in humans, which manifests itself as skin injury resulting from an excessive accumulation of uroporphyrin. In his editorial about the study, Crispin B. Taylor of the American Society of Plant Biologists referred to this plant as a vampire plant because its phenotype is similar to what is observed in photosensitized patients afflicted with porphyria. To date, plants with other natural dysfunctions in their porphyrin pathway, such as a deficiency in protoporphyrinogen oxidase as in variegate porphyria, are yet to be identified.

The first herbicide that included a protoporphyrinogen-oxidase inhibitor, nitrofen, was introduced in 1964, but the mechanism of action of these herbicides perplexed scientists for many years. They cause rapid photobleaching and light-dependent desiccation of foliage, resulting in leaf cupping, crinkling, bronzing and necrosis. The lesions on the foliage are caused by a loss of membrane integrity that leads to cellular leakage. Other physiological responses include inhibition of photosynthesis; evolution of ethylene, ethane and malondialdehyde; and, finally, bleaching of chloroplast pigments. Protoporphyrinogen-oxidase inhibitors are known to cause temporary injury to the foliage of treated crops they were designed to protect, but these plants recover rapidly because they quickly degrade the herbicide.

The light-dependent activity of protoporphyrinogen-oxidase inhibitors was recognized by 1969, but their actual mechanism of action eluded scientists for decades. In the late 1980s, the French research group of Michel Matringe and his colleagues found that the loss of membrane integrity results from the accumulation of a photodynamic pigment, which was soon identified as protoporphyrin (see Figure 7). Although this result pointed to the inhibition of a biochemical step downstream from the point of synthesis of protoporphyrin, none of the enzymes tested proved to be the target. The same research group, in collaboration with experts in porphyrin biochemistry, realized how similar this effect is to the accumulation of protoporphyrin in human deficiency of protoporphyrinogen-oxidase activity (variegate porphyria) and reported that these herbicides indeed targeted the chloroplastic enzyme. The apparent paradox of inhibition of an enzyme leading to the accumulation of its catalytic product is explained by the altered subcellular compartmentalization of porphyrin intermediates. As with humans with variegate porphyria, inhibition of protoporphyrinogen oxidase induces an uncontrolled accumulation of protoporphyrinogen, which in plants leaks out of the chloroplasts’ outer membrane into the cytoplasm, where it is converted into the highly photodynamic protoporphyrin. In the presence of light, protoporphyrin generates highly reactive singlet oxygen species that cause lipid peroxidation of the relatively unprotected plasma membrane.

Resistance to protoporphyrinogen-oxidase–inhibiting herbicides in weeds has been slow to evolve, but resistant biotypes of water hemp (Amaranthus tuberculatus) have recently been identified. These biotypes have broad levels of cross resistance to several classes of protoporphyrinogen-oxidase inhibitors. Resistance has been associated with reduced accumulation of protoporphyrin, which was accompanied by reduced membrane damage. Resistance was characterized as an incompletely dominant trait on a nuclear gene called PPX2L. This gene encodes the mitochondrial protoporphyrinogen-oxidase homolog, which is not the primary target of these inhibitors. However, this gene possesses an amino- terminal extension, which leads to dual targeting of the gene product toward both plastids and mitochondria.

Instead of a usual single-point mutation resulting in the substitution of one amino acid for another, the research group of Patrick J. Tranel at the University of Illinois discovered that the herbicide-resistant PPX2L gene has a codon deletion for glycine at a critical position (see Figure 8). This improbable mutation may have occurred as a result of the slippage of DNA polymerase in an array of microsatellite repeats. One of us (Franck E. Dayan) and his colleagues established that the deletion did not affect the affinity of protoporphyrinogen or the FAD content, but it decreased the catalytic efficiency of the enzyme. The suboptimal efficiency was compensated for by a change in the binding mode of the herbicides and a significant increase in their affinity for protoporphyrinogen oxidase. The seemingly innocuous deletion of a glycine in fact altered the conformation of protoporphyrinogen oxidase significantly because this amino acid residue plays a key role in stabilizing the helix.

One Ring

I saw eternity the other night
Like a great ring of pure and endless light,
All calm as it was bright,
And round beneath it time in hours, days, years,
Driven by the spheres,
Like a vast shadow moved in which the world
And all her train were hurled.
— Henry Vaughan, The World

Nature’s propensity to embrace specific molecules (amino acids, nucleic acids, fatty acids and carbohydrates) as the building blocks of structures with grander purposes seems to be consistent with its usage of the porphyrin ring. The unique metabolic pathway producing protoporphyrin, the master ring from which the other “rings of life” are derived, is ubiquitous and appeared early in the evolution of life. The biological importance of these molecules dictates that any disturbance of their synthesis, and particularly of the terminal step that forges the “One Ring to rule them all,” has catastrophic consequences in all aspects of life.