Binding to Cytochrome Oxidase and the Mysterious Cyanhemoglobin
by Steven Curry, M.D.
Banner – University Medical Center Phoenix
University of Arizona College of Medicine – Phoenix
Every toxicology fellow learns about cyanide. But not all of what they learn is necessarily correct. As a resident, I was taught that the CN– ion initially bound to ferric iron (Fe3+) in cytochrome oxidase to stop electron transport and inhibit oxidative phosphorylation. But, is this true, given cyanide’s alkaline pKa? I had also read that a person suffering from cyanide poisoning was bright pink or red and had an elevated venous oxygen content. But then shortly after residency, I read that CN– not only bound to cytochrome oxidase but also combined with hemoglobin to form a compound named cyanhemoglobin that was incapable of transporting oxygen. This supposedly causes an unexplained decrease in oxygen saturation of hemoglobin (percent saturation gap). I wondered which was more important.
Here we will examine some aspects of the toxicomythology of cyanide poisoning, perhaps best-called cyanomythology. There are quite a few myths, and it would take countless pages to cover them all in any detail. So I have chosen two of the least recognized myths to discuss today: cyanide’s binding to cytochrome oxidase and the enigmatic cyanhemoglobin.
Forms of Cyanide in Industry and in vivo
We generally encounter cyanide in three major forms, 1) solid cyanide salts, 2) hydrogen cyanide (HCN) gas or liquid, and 3) cyanogens – compounds that are metabolized to cyanide during or after absorption. Cyanogens include various cyanide glycosides in food and “natural” products, such as amygdalin, nitriles (e.g. acetonitrile) found in industry, laboratory settings, and some commercial products, and the drug, nitroprusside. We aren’t going to talk much about cyanogens today. Let’s first focus on cyanide salts and HCN.
Occupational or hazmat encounters with cyanide that result in serious poisoning commonly result from exposures to HCN. While HCN gas can be stored in a cylinder (commonly as a calibration gas) or dissolved in water to make a hydrocyanic acid solution (prussic acid), what occurs most commonly is the accidental formation of HCN during the use of cyanide salts. Capital punishment with HCN using gas chambers in the U.S. began in the 1920s and remains a legal option today in a few states; this includes my own state of Arizona where the last US execution by cyanide occurred in 1999. HCN gas intentionally is formed by remotely dropping KCN or NaCN into a container of sulfuric acid. The reaction with KCN is as follows:
2 KCN + H2SO4 → K2SO4 + 2 ↑HCN
But take a look at the HCN dissociation curve, below. The Y-axis is the percent of cyanide present as HCN (much of which would off-gas) in an aqueous solution of a cyanide salt, and the X axis is the solution’s pH.
It is obvious that in H2SO4 at pH < 1 (off the chart to the left), all that KCN in the gas chamber will go to HCN. But the pKa of HCN is about 9.2 or so. In fact, look at pH 7. If we added KCN to a large volume of pure water, we would also find about 100% as HCN. That’s right. Cyanide salts in water at pH 7 off-gas HCN.
KCN + HOH → ↑HCN + KOH
As more and more KCN is added to water, more KOH, a strong base, is formed, and the pH of an aqueous KCN solution becomes more alkaline. HCN, a weak acid, does not dissociate as much and off-gasses until the solution’s pH exceeds about 11. But plenty of HCN can be released, and this is why it is generally unwise to mop up or attempt to hose off spilled cyanide salts with water. The ingestion of cyanide salts, through the formation of the hydroxide, can produce gastric irritation and a corrosive injury.
Aqueous solutions of cyanide salts must have pHs maintained greater than 10.5 to 11 to prevent significant off-gassing of HCN. If you happen to visit electroplating or precious metal reclaiming facilities, you will see lots of metal cyanide salts in solution, and the pHs of those solutions are usually kept quite high. Adding cyanide salts to water before the pH has been raised can lead to a disaster, and much more so if mistakenly mixed with acid. (Note: there are methods of electroplating using metalocyanide compounds at acidic pHs, especially gold, but I will not discuss those.)
Cyanide Binding to Cytochrome Oxidase
With this background it should now be apparent that at a physiologic pH the great majority of absorbed cyanide in our body will be circulating in the form of HCN, whether we ingest cyanide salts, are sprayed with acrylonitrile, or inhale HCN. And that should make you suspect that it is not CN– that binds to cytochrome oxidase, but HCN. Indeed, this is the case. The center of cytochrome a3, where cyanide binds, is quite hydrophobic and prefers initially to bind to neutral species (e.g., HCN, H2S, HN3). Cooper and Brown authored a nice review that fellows might like to keep in their files.1
For toxins with acidic or neutral pKas, (e.g. azide, sulfide, formate) that bind to cytochrome oxidase, metabolic acidosis will increase the neutral, protonated species and, therefore, increase cytochrome binding. This is the main reason I am aggressive in correcting metabolic acidosis in my patients with methanol poisoning. But this isn’t true for cyanide with its alkaline pKa. HCN remains the main species in all pH ranges compatible with life.
Now we come to the issue of where HCN initially binds. Many sources, including this author, have stated that cyanide initially binds to the ferric iron of cytochrome a3 in cytochrome oxidase. Let’s look at this more closely by examining the figure, below.
The top of the figure is the intermembrane space between the inner mitochondrial membrane (cristae) and the outer membrane. The bottom is the matrix where the TCA cycle occurs. In the example above, NADH unloads electrons (green arrows) onto complex I, while succinate from the TCA cycle unloads electrons onto complex II. Both I and II shuttle electrons to Q (ubiquinone) and then onto complex III. Complex III shuttles electrons onto cytochrome c which sits on the outside of the inner membrane. Cytochrome c gives up electrons to complex IV. Because complex IV oxidizes (removes electrons from) cytochrome c, it is known as cytochrome oxidase or cytochrome c oxidase. Complex IV, in turn, puts the electrons onto oxygen, the final electron acceptor, to make water. The energy released as electrons move down the chain is used to pump protons from the matrix into the intermembrane space. Thus, the matrix is relatively negative to and has a higher pH than the intermembrane space, like a battery.
Protons in the intermembrane space desperately want to move down their electrochemical gradient into the matrix. They do so by moving through a pore in ATP synthase, which causes the enzyme to synthesize ATP. This is oversimplified oxidative phosphorylation in a nutshell.
Let’s look at complex IV in a bit more detail.
Complex IV comprises 13 protein subunits (3 coded by mitochondrial DNA) and contains several moieties that undergo oxidation and reduction as electrons move through it (red arrows). The hexagon, top left, is cytochrome c, which undergoes oxidation when it unloads electrons onto two copper atoms in cytochrome oxidase, termed CuA. From CuA electrons flow to the iron in the center of cytochrome a. And next in line is what is termed the binuclear center of cytochrome a3. The name, binuclear center, refers to the fact that both an iron in cytochrome a3 and yet another copper atom (CuB) participate in redox reactions in a coordinated manner, and it is from the binuclear center that electrons move onto oxygen to form water.
HCN initially binds somewhere to the binuclear center of cytochrome a3 to very rapidly inhibit cytochrome oxidase. A requirement for cyanide’s binding is that an electron is located on cytochrome a or CuA. In vitro, rapid cyanide binding completely inhibits cytochrome oxidase activity within 50 ms, a time typical of the initial electron transfer from cytochrome c to CuA/cytochrome a. But where does HCN initially bind? Let’s go yet deeper and look at another cartoon to examine the various redox states of the binuclear center.
Things are a bit more complicated than simple ferrous or ferric iron. Iron’s state of oxidation ranges from Fe2+ to Fe4+, and copper bounces between Cu+ and Cu2+. HCN’s binding to either copper or iron could inhibit electron transport and oxidative phosphorylation. So, which is it? Fellows usually read or are taught that it is binding to ferric iron. But, in fact, in 1965 Camerino and King performed experiments that suggested two binding sites for cyanide and suggested one of them could be copper.2 And they cited yet an earlier 1961 paper in support of this.3 For those of us who clearly recall life in 1961, The Flintstones had only been on the air for about a year.
Jump 33 years to 1994. Wilson and colleagues reported that HCN’s initial binding is not to iron, but to CuB2+, and others had and have come to similar conclusions.4 Wilson found that over longer periods of time cyanide binding to the iron of oxidized cytochrome a3 does, indeed, occur, either by transfer of cyanide from CuB, or by forming a bridge between the two metals (possibly with oxidation of HCN; Fe – CN – Cu), or possibly by the binding of a second HCN molecule. But they found forming a ligand to iron in cytochrome a3 was irrelevant to the onset of inhibition by cyanide.
HCN’s formation of ligands with metals such as copper and iron occurs through coordination bonds, in which HCN provides both electrons. I have two medicinal chemistry gurus, one being my good friend, Ed Boyer. Ed is different than most of us in that he sees atoms as electron probability density functions and speaks of “soft sulfur atoms” and other strange phenomena. When we have discussed coordination bonding to metals, Ed has reminded me that although these bonds are covalent, “they are weird” and depend more on fitting orbitals together physically rather than just dealing with outer electron shells. Although covalent, cyanide’s coordination bond to the binuclear center is reversible, which explains why we can treat cyanide poisoning with antidotes that tie up or metabolize cyanide.
The Enigmatic Cyanhemoglobin
Thus far we have recognized that 1) cyanide present in blood and cells exists mainly as HCN, and 2) HCN initially binds to Cu2+ in the binuclear center of cytochrome a3 of cytochrome oxidase. But what about the claim that cyanide binds to ferrous iron (Fe2+) of hemoglobin to form something named cyanhemoglobin? As I write this in May 2018, I am looking at the cyanide section of UpToDate®, which states:
“Although cyanide has a primary affinity for ferric (Fe3+) iron, a small amount may bind to the ferrous (Fe2+) iron of hemoglobin, forming cyanohemoglobin, which is unable to transport oxygen, thereby further exacerbating tissue hypoxia.”
Going back 32 years, this is from a 1986 review on cyanide poisoning from a widely read medical journal:5
“Some cyanide binds to the ferrous (Fe2+) iron of normal hemoglobin. This cyanhemoglobin cannot transport oxygen. The presence of significant amounts of cyanhemoglobin may be indicated by a decrease in the measured arterial %O2 saturation (measured directly with a co-oximeter), while the calculated %O2 saturation (derived from a nomogram using measured PO2 and total hemoglobin values) remains normal.”
If this wasn’t intriguing enough, in 1990 Poisindex® stated that cyanhemoglobin also shifted the oxyhemoglobin dissociation curve to the left.
These are examples of several authors who have described ferrous cyanhemoglobin. Let’s try to track cyanhemoglobin down and understand its relevance to poisoned patients.
But first a caution. We know that cyanide loves to form a ligand with ferric methemoglobin to form cyanmethemoglobin. One must be careful when reviewing older literature regarding cyanhemoglobin. Beginning long ago and continuing to present day, our laboratories convert all hemoglobin species (oxyHb, reduced Hb, metHb, COHb, etc.) to cyanmethemoglobin when determining total hemoglobin concentration such as found in a CBC. And the term, cyanhemoglobin, has been used synonymously with cyanmethemoglobin for over 100 years. We must be careful to distinguish between cyanhemoglobin as cyanmethemoglobin, and the alleged cyanhemoglobin existing as a ligand between cyanide and ferrous hemoglobin. With that in mind, let’s begin.
In 1871, the year Germany became a nation, William Thierry Preyer published his book, Die Blutkrystalle.
Most all references regarding the formation of cyanhemoglobin begin here or with Preyer’s contemporaries dating back another decade or so. Preyer and others were quite interested in hand spectroscopy of various pigments formed in blood in vitro. The examples of spectra from his book, below, are the sorts he was observing. Preyer would take defibrinated blood and hemolyze it, typically by diluting it 1:1000 in water, and examine the spectra after the addition of various acids, alkali, salts, and gases.
He found that if he shook a tube of diluted hemolyzed blood in air he could see two lines on his spectroscope, which he considered oxidized heme. If he mixed dilute hemolyzed blood with a KCN solution or with HCN (as gas or prussic acid), nothing happened initially, and his spectroscopic reading remained unchanged. However, if he let the solution sit at room temperature for hours, a brown-orange color developed. Subsequent spectroscopy showed the two lines of oxygenated blood had disappeared, having been replaced by a washed-out absorption band. Shaking in air would not restore the oxygenated state, and adding a reducing agent had no effect, either.
If he performed the same experiment (hemolyzed dilute blood with KCN or HCN) and heated the blood, the reaction took place rapidly. There was nothing about anyone with cyanide poisoning possessing this pigment in their blood, whatever it may have been, but which we now suspect was cyanmethemoglobin and/or a hemochromogen (described later). Adding KCN or HCN to diluted hemolyzed blood could certainly cause significant pH changes, denature proteins and cause oxidant stress.
In his 1913 text on the use of the spectroscope, Emil Rosenberg reported that Preyer’s observation on the conversion of oxygenated ferrous hemoglobin to cyanhemoglobin could not be demonstrated in the blood of poisoned animals, and that
“the same negative result appears in the single observation recorded concerning the spectrum examination of the blood of man after poisoning by prussic acid. H. Siegel (Leipzig) . . . found in this case, the blood dark, wholly fluid; in thin layers it appeared cherry-red, and the two absorption-bands of O2-Hb presented themselves in all distinctness.”
Thus, by 1913 it was recognized that formation of cyanhemoglobin in diluted hemolyzed blood mixed with cyanide and allowed to sit at room temperature for hours didn’t appear to be an in vivo phenomena.
Jumping forward a few years, William Christopher Stadie was the first to describe low O2 content in arterial and venous blood as an explanation for cyanosis seen in victims of the 1918 influenza pandemic. He described oxygen therapy for influenza patients and reported improvement or resolution of cyanosis. This was big news at the time.
But Stadie has also been occasionally cited regarding cyanhemoglobin. In 1920, Stadie published a paper on the measurement of hemoglobin in blood. He desired to convert all hemoglobin to what he called cyanhemoglobin (our cyanmethemoglobin) for measurement of total hemoglobin concentrations, just as our laboratories do today (J Biological Chem 41:237-241).
Citing Preyer’s work, Stadie wrote that dilute KCN could also change ferrous hemoglobin to cyanhemoglobin in hemolyzed blood, but this occurred in vitro very slowly at room temperature. In his method he hemolyzed blood with water and then added 5 mg KCN to the hemolysate, producing a cyanide concentration of about 37 mg/L. It was this solution that had to sit for hours before it would convert partially to cyan(met)hemoglobin. Stadie reported that the spectra of cyanide mixed with methemoglobin was identical to the spectra of cyanide mixed with normal blood and heated for 30 minutes, indicating that both procedures produced what we would call cyanmethemoglobin. However, there was nothing described about forming a ligand between cyanide and ferrous hemoglobin in vivo.
In 1963, Jay Arena, MD published the first edition of his book, Poisoning: Chemistry, Symptoms, Treatments. Dr. Arena served as president of the American Academy of Pediatrics and led the charge to get the FDA to establish the National Clearinghouse for Poison Control Centers in 1957.
Arena wrote that cyanide slowly combines with hemoglobin to form small amounts of a stable, non-oxygen-bearing compound named cyanhemoglobin, though he clarified that cyanhemoglobin did not contribute to morbidity or death. Dr. Arena was either extrapolating from in vitro experiments initially reported by Preyer and others in the 19th century, or describing the fact that some cyanide would bind to the small amount of methemoglobin normally present in red cells. If he was referring to something else, we won’t know since none of his statements were referenced.
In 1974, Yacoub and colleagues published a paper that was regularly cited in the 1980s and 1990s in support of the existence of ferrous cyanhemoglobin.6 In a translation my friend Alan Hall was kind enough to provide to me long ago, they wrote:
“Although the bond of cyanide to hemoglobin is weak, it exists nevertheless, producing cyanhemoglobin, which explains the copious amount of cyanide in the red cell and the spleen.”
Unfortunately, no references were cited to support this statement.
In 1980, Vesey reported on incubation of red cells in cyanide solutions and found that the initial increase in red cell cyanide levels was not accompanied by a fall in oxygen-carrying capacity, providing evidence against cyanhemoglobin in intact erythrocytes.7
Prolonged incubation did decrease oxygen content slightly, but the cyanide levels required were so high a patient would have been long dead. And we don’t know to what cyanide was presumably binding.
Next came McMillan and Svoboda, who published a paper on the importance of erythrocytes in detoxifying cyanide.8 They wrote that cyanide was concentrated in the red cell by binding to hemoglobin and cited Anson and Mirsky when writing:
“Cyanide binds less strongly to reduced heme (Anson and Mirsky, 1928) but more ferrous hemoglobin is present, so that cyanide probably binds initially to unaltered hemoglobin.”
Might Anson and Mirsky’s 1928 paper in Journal of General Physiology9 give us the data we have been seeking? Perhaps, finally, we are getting somewhere! Keep your fingers crossed.
Sadly, Anson and Mirsky never described a ligand between cyanide and ferrous hemoglobin. I’ll bet you aren’t surprised. Rather, they described how cyanide could form a hemochromogen. A hemochromogen is usually created through denaturation of hemoglobin (e.g., heat, alkali, acid) resulting in a heme group in which small ligands such as cyanide bind to the coordination site where the globin of hemoglobin would normally bind. But, of course, such sites are unavailable for binding to ferrous hemoglobin in vivo.
A couple more points that would make Anson and Mirsky’s paper even more clinically irrelevant: 1) the ferrous hemochromogen had to be studied in anaerobic conditions lest the iron immediately oxidize to Fe3+, and 2) these hemochromogens were created in vitro in 1% sodium carbonate at pH about 11.6. These are not conditions we will encounter in the blood of living human beings very often.
In 1991 Henry Patrick and I published a paper on the lack of evidence for a percent saturation gap in cyanide poisoning.10 Briefly, we incubated arterial and venous blood samples from ICU patients in 0, 6, 12, or 25 mg/L cyanide at buffered physiologic pH. We then performed multiwavelength co-oximetry on samples to measure hemoglobin species and found nothing to suggest a “saturation gap”, any change in oxyhemoglobin levels, or any shift in the dissociation curve.
It’s 27 years later and 105 years after Rosenberg informed us that cyanhemoglobin is not found in vivo, and authors are still writing about cyanhemoglobin in patients with cyanide poisoning. The enigma is no longer cyanhemoglobin but the fact that persons still write about it.
As a quick summary, let’s examine some common statements regarding cyanide poisoning in human beings and see how we would now judge their validity.
- Cyanide toxicity mainly results from cyanide ions (CN–) binding to cytochrome oxidase.
False. HCN is the main species that binds to cytochrome oxidase.
- Cyanide initially binds to ferric iron of cytochrome oxidase.
False. HCN appears to initially bind to Cu2+ in the binuclear center of cytochrome a3 to immediately inhibit cytochrome oxidase. Binding to the iron of heme does eventually occur but is not required for inhibition of electron transport.
- Cyanide binds to ferrous hemoglobin to produce meaningful amounts of cyanhemoglobin, a compound incapable of transporting oxygen.
- Cyanide produces a percent saturation gap.
- Cyanide shifts the oxyhemoglobin dissociation curve to the left.
That’s enough cyanomythology for today. Please leave feedback regarding the depth and length of what we covered. Feynman noted that “. . . everything is interesting if you go into it deeply enough . . .”, and that certainly holds true for toxicology. But if you want shorter and more superficial discussions, tell me. Or we can go deeper.
Finally, if any of you are not toxicology fellows, but have actually read to the end of this post, you may have been born to be a medical toxicologist. You might consider a career redirection.
Andrew Miller says
I for one loved the depth and would absolutely geek out on more. Fantastic post.
Steve Curry says
Andrew – thanks so much for the comment. I wish I could get about 50 more feedback comments to get a general impression of what fellows and others want. This is the sort of stuff we teach our fellows, but not sure that’s what other fellows are interested in learning about.
Scott Weingart, MD FCCM says
As a non-toxicologist I also think the depth is perfect. It is definitely beyond me, but that is where learning occurs. The secret to a well rated lecture (or blogpost) is to give 90% review and 10% new. You will get mass appeal and high scores. Those kind of lectures are garbage for anyone that truly wants to expand their knowledge base. Keep up this level of blogpost–please!!!!!!!!!!!!!
Steve Curry says
Scott, thanks for the feedback. This is very helpful. While trying to write for toxicology fellows, I also want to be sure that other physician specialists and those in training may find it helpful.
Marco Sivilotti says
Feynman also said that “Science is the belief in the ignorance of experts…” Thank-you for this deep dive into the Science of such a simple molecule, HCN, and nicely illustrating the wisdom of the quote! Notable for its absence is any mention of the underlying premise of the 3-part Lily antidote kit… I am also left wondering about the process/chemistry by which modern automated CBC analysis instruments go about generating cyanomethemoglobin (e.g. lysed or intact cells, under what pH and HCN/CN- concentrations?). As for future topics, maybe CO, another very poorly understood but much maligned micromolecule??
Ole Bjørsvik says
Could you explain the hydrogen arrows in the yellow circle a bit better. They don’t make full sence for me. At least not the one on the right side.
Steven Curry says
The figure is simply showing the steps at which 2 protons are “pumped” from the matrix into the intramembrane space for each pair of electrons. If we were to go into more detail, what actually happens is that 2 electrons donated from cytochrome c make their way to cytochrome a3 of the binuclear center where O2 is bound and is reduced to the peroxyform (O2 2-). Then 2 more electrons come down the line and convert the peroxy form to 2 molecules of water. This entire process results in consumption of 4 protons by the O2 to make 2 H2O while simulataneously 4 other protons are pumped out of the matrix.
Steven Curry says
Oops. Itramembrane space is supposed to be intermembrane space.
As a tox fellow, i’ve read a lot about cyanide but this one is the best :))
Another tox fellow here – this post was fantastic. Good mix of dogmalysis and historical perspective – this kind of information is really difficult to work into a coherent story if you go looking on your own, so I appreciate how you’ve presented it here.
Steven Curry says
Thanks Justin. The goal is to provide information to toxicology fellows that is important (for practice and for boards) but not well-explained, if at all, in standard references. I think Howard is formatting the next post, and we will go back in history a bit more in that one.
Jeff Suchard says
Steve, this was great stuff! This will definitely influence how and what I teach my students and residents about cyanide.
I also have some questions about the redox reactions occurring at the binuclear center, in the figure with the yellow circle. Of course, you were focusing on the oxidation states of iron and copper, so this figure doesn’t make specific accounting of all the oxygen and hydrogen atoms, and I want to be sure I’ve balanced all the reactions correctly. I believe the following 3 observations are correct:
– Each addition of an electron appears to also imply the addition of a proton [not depicted]
– The two reactions between the 6-o-clock and 10-o-clock positions each yield a water molecule [not depicted]
– There is no chemical reaction at the binuclear center between the 2-o-clock and 4-o-clock positions;
rather, this just happens to be the phase where a proton is pumped into the matrix. And if so, I am puzzled why there need to be separate boxes at the 2- and 4-o-clock positions [This appeared also to have confused a previous commenter]
The other thing puzzling me is whether there needs to be conservation of total amount of positive oxidation state of the iron and copper in the reactions, or if I am mentally imposing rules that do not need to occur in reality. Given that this total is NOT conserved, as depicted in the figure, I am presuming there is no such requirement. [My Physical Chemistry skills are rusty (Fe2O3-H2O : terrible pun intended)]
Steven Curry says
Hi Jeff. As you noted, the figure was extremely simplified and meant to emphasize the various valencies of iron and copper during the movement of electrons through the binuclear center onto water. I am sending you a special present – my favorite condensed read on oxidative phosphorylation and mitochondrial bioenergetics. In fact, I recommend to everyone who wants to read in more depth on the topic the book, Biogenergetics 4, by David G Nicholis. I have read each version, beginning with Biogenergetics 2, and the book is outstanding. The book contains a beautiful illustration for the reaction between cytochrome oxidase and oxygen, including proton pumping steps. And each step is balanced, beginning with 4 electrons moving from cytochrome a into the binuclear center. The book is about 430 pages. Enjoy!
Steven Curry says
Jeff, I replied last night and saw it posted, but my reply has disappeared. Indeed, the cartoon of the binuclear center was mainly to illustrate the oxidation states of iron and copper while electrons and protons were flying around. It certainly is not complete or balanced. I have sent you a wonderful book, Bioenergetics 4, by David Nicholis. I’ve read Bioenergetics 2, 3 and 4 and all are outstanding and contain the exact figures, including balanced electrons and protons during various redox states of the binuclear center, that you seek. Bio 4 is up to about 430 pages, but great reading.
Scott Weingart, MD FCCM says