The Iron-Oxygen Bond in Oxyhemoglobin

Pastel drawing of the hemoglobin molecule by Roger Hayward, 1964.

Part of the beauty of studying the life and work of Linus Pauling is that doing so often affords the opportunity to look at how the science of today has developed from questions that were once unanswered and widely debated. One such question was how hemoglobin, the protein in red blood cells, binds to and releases oxygen as it is inhaled and carried to the body’s tissues.

In 1959, Max Perutz used x-ray crystallography to obtain an image of oxyhemoglobin, a hemoglobin protein bound to an oxygen molecule. This was a major breakthrough in many ways. For one, it allowed chemists to observe an image of a three-dimensional protein found in humans for the first time. The imagery also provided tantalizing hints about the specific chemistry that might explain oxyhemoglobin’s structure.

Unfortunately, creating an image of the molecular structure didn’t solve everything, as Perutz himself would reflect in a 1978 article, “Hemoglobin Structure and Respiratory Transport.”

We were like explorers who have discovered a new continent, but it was not the end of the voyage, because our much-admired model did not reveal [hemoglobin’s] inner workings.

The Joseph Weiss Medal, which commemorates his work as a radiation chemist.

The Joseph Weiss Medal, which commemorates his work as a radiation chemist.

Perutz’s work naturally incited biochemists to further explore the structure of hemoglobin. While it was known that the oxygen-carrying heme group in hemoglobin is composed of nitrogen, carbon, and an oxygen-binding iron, there was much debate over what kind of bond could cause the union and dissociation of these elements.

In 1964, Joseph Weiss, a professor at Newcastle University in England, attempted to answer the question of what specific bond forms between iron and oxygen. Weiss’s conclusions were published in a 1964 article, “Nature of the Iron–Oxygen Bond in Oxyhæmoglobin”.

According to Weiss, the iron in hemoglobin would need to be in the ferric state (iron with an ionic charge of +3) in order to account for hemoglobin’s behavior in oxygen transport.  He believed that ferric iron would also explain hemoglobin’s spectroscopy (the wavelengths of light reflected by a  molecule). Pauling, however, disagreed with Weiss.

Pauling, Max Delbruck and Max Perutz, 1976.

Interestingly, Pauling had been looking into the subject of the iron-oxygen bond in hemoglobin since 1948, when he presented a paper titled “The Electronic Structure of Hemoglobin” at a symposium in Cambridge, England. Pauling’s presentation considered advances in x-ray diffraction and quantum mechanics to propose a structure for the heme group in the protein. Unlike Weiss, Pauling believed that the iron-oxygen bond in oxyhemoglobin would require ferrous iron (an iron with an ionic charge of +2) to form a double bond (a bond involving two electrons) with oxygen as it was being transported throughout the body.  Weiss’s paper did little to change Pauling’s mind on the subject.

In 1964, Pauling wrote “Nature of the Iron–Oxygen Bond in Oxyhæmoglobin,” a direct response to Weiss’s article with the same title. In it, Pauling stated

I conclude that oxyhæmoglobin and related hæmoglobin compounds are properly described as  containing ferrous iron, rather than ferric iron, that their electronic structure involves essentially the formation of  a double bond between the iron atom and the near-by oxygen atom in  oxyhæmoglobin

Pauling’s interest in the components of blood had emerged early on in his career. In 1948 he suggested using hemoglobin to test his earlier ideas about bonds that had remained unexplored, as the structure of the protein had been hitherto not fully understood. This was a pursuit that he in which he strongly believed: in his Cambridge talk, “The Electronic Structure of Hemoglobin,” he had concluded that

even the great amount of work that would be needed for a complete determination of [hemoglobin’s] structure, involving the location of each of the thousands of atoms in its molecule, would be justified.

Many years later, in his 1992 article “The Significance of the Hydrogen Bond,” Max Perutz stated that that Pauling’s words, as published in 1949, were among the inspirations propelling his own work a decade later.

“The Electronic Structure of Hemoglobin” wasn’t the only publication by Pauling that inspired Perutz. In 1970, he used Pauling’s “The Magnetic Properties and Structure of Hemoglobin” to further his own study of the structure of hemoglobin, work which finally led to the discovery that the iron-oxygen binding in hemoglobin depends on the electronic spin transition of the iron atom.

Essentially, Perutz found that when the ferrous iron in hemoglobin is in a low spin state, its higher d-orbitals are unoccupied by electrons, which allows oxygen to form a bond with iron. In a high spin state, the electrons in ferrous iron are occupying all d-orbitals in the atom and oxygen remains unbound.

This suggests that the more likely structure for hemoglobin involves a single bond between iron and the oxygen molecule, not the double bond that Pauling had proposed in 1948 and again in 1964.  But Pauling was correct with respect to the presence of ferrous iron in the compound, and he had been able to make this determination before any crystallographic pictures were available to him.


Pauling’s First Hemoglobin Publications: Understanding Oxygen Binding

Pastel drawing of the hemoglobin structure, by Roger Hayward. 1964.

“You know, hemoglobin is a wonderful substance. I like it. It’s a red substance that brings color into the cheeks of girls, and in the course of my hemoglobin investigation I look about a good bit to appreciate it.”

– Linus Pauling, March 30, 1966

Seventy-five years ago, in 1935, Linus Pauling began publishing his research on the protein hemoglobin with a set of papers titled “The oxygen equilibrium of hemoglobin and its structural interpretation” appearing in Science and the Proceedings of the National Academy of Science .

In the fall Pauling extended this work and began collaborating with newly minted Caltech Ph. D. Charles Coryell, on the problem of the binding of oxygen to hemoglobin in the formation of the compound oxyhemoglobin. In April 1936, the duo published a paper specifically devoted to the subject, “The magnetic properties and structure of hemoglobin, oxyhemoglobin, and carbonmonoxyhemoglobin,” an important article which appeared in PNAS.

In order to better understand this early hemoglobin work, it is important to first discuss some of the basics of the hemoglobin molecule. Hemoglobin is a major protein component in the cytoplasm of red blood cells, and is made up of two distinct parts – the heme and the globin. Its primary function is to facilitate gas exchange: it picks up oxygen in the lungs, carries it to the tissues, and returns to the lungs in order to expel the carbon dioxide produced in the tissues.

There are four hemes per hemoglobin molecule, and each is made up of a single iron atom surrounded by a porphyrin ring. Each heme has the ability to bind to a single oxygen dimer, therein giving hemoglobin the capacity to bond with four molecules of O2. The globin is the main protein component of the molecule. Carbon dioxide, rather than competing with oxygen for a binding site at the heme, instead binds to the globin.

Charles Coryell and Linus Pauling. 1935.

In their 1936 paper, Pauling and Coryell tackled the question of how oxygen binds to hemoglobin by looking at the molecule’s magnetic behavior, using an experiment involving bovine blood and magnets.  In a 1976 interview, Pauling provided this description of their experimental design.

It occurred to me that the same magnetic methods that we had been using to study simple compounds of iron, in order to determine the bond type, could be used to study the hemoglobin molecule. One of my students, Charles Coryell, and I, then got some blood, cattle blood, and put it into an apparatus. It consisted of a balance, which we had fitted out in such a way that a wire was suspended from one arm of the balance through a hole in the base of the cabinet, and held a tube. This tube was placed between the poles of an electromagnet. We filled it with blood, oxygenated blood, and balanced it to measure its weight. Then we passed an electric current through the coils of wire and the apparent weight changed.

From the experimental results, the pair found that oxyhemoglobin contains no unpaired electrons, although free oxygen molecules contain two, and each heme contains four. This was something of a surprise as, quoting from the paper,  “It might well have been expected, in view of the ease with which oxygen is attached to and detached from hemoglobin, that the oxygen molecule in oxyhemoglobin would retain these pair of electrons.”

In spite of this possibly more intuitive expectation, Pauling had earlier theorized that oxygen binds to hemoglobin covalently, a prediction which the experiment confirmed. Indeed, it was found that “the oxygen molecule undergoes a profound change in electronic structure on combination with hemoglobin,” and binds to the iron atom in the heme covalently.

Pastel drawing of Hemoglobin at 100 angstroms, 1964.

This was, however, only one of the striking discoveries that surfaced out of this research.  In a deoxygenated hemoglobin molecule, the bonds between iron and the four porphyrin nitrogen atoms surrounding it are ionic. Nonetheless, upon the binding of oxygen, these bonds become covalent, a rather dramatic change. Pauling and Coryell were keen to point this out:

It is interesting and surprising that the hemoglobin molecule undergoes such an extreme structural change on the addition of oxygen. Such a difference in bond type in very closely related substances has been observed so far only in hemoglobin derivatives.

Clearly something of consequence was being observed.  In their conclusion, the authors noted as much.

It is not yet possible to discuss the significance of these structural differences in detail, but they are without doubt closely related to and in a sense responsible for the characteristic properties of hemoglobin.

Linus Pauling’s work with hemoglobin continued on and off until his death in 1994, and led to a number of important discoveries – most prominent among them the molecular basis of sickle cell anemia. For more information on Linus Pauling’s hemoglobin research, please visit the website It’s in the Blood! A Documentary History of Linus Pauling, Hemoglobin, and Sickle Cell Anemia.