The Nobel Prize in Physiology or Medicine in 1931 was awarded to Otto Heinrich Warburg “for his discovery of the nature and mode of action of the respiratory enzyme.” The Nobel Lecture presented by Warburg on December 10, 1931 “The oxygen-transferring ferment of respiration” shed light on how the complicated mechanism of oxidation and reduction observed during respiration in every living cell involved BOTH OXYGEN AND IRON .
The term “Warburg Effect” used frequently in oncology was coined by Efrain Racker in the early 1970s as an alternative description for aerobic glycolysis. In the 1920’s Warburg and his colleagues observed that tumor cells could ferment large amounts of glucose even in the presence of oxygen. Since glycolysis is an anaerobic fermentation process that requires no oxygen, the process used by tumor cells to produce energy was referred to as aerobic glycolysis .
In his paper “THE METABOLISM OF TUMORS IN THE BODY” published in 1926, Warburg et al. made this very important statement “We assume it is understood that tumor cells obtain the energy required for their existence in two ways: by respiration and by fermentation . The fact that tumors can also survive by respiration explains why cancer cells love iron.
Iron, Oxygen & Mitochondria: Warburg Revisited
All living organisms on earth depend upon iron as an essential element, from all phyla of bacteria , to plants, animals and humans . Cancer cells are no exception. Iron metabolism is often highly dysregulated in tumor cells. The altered expressions of iron-related proteins usually result in a relatively high level of intracellular iron that support various essential biological functions in DNA synthesis and repair, cell cycle regulation, angiogenesis, metastasis, tumor microenvironment and epigenetic remodeling .
Why is iron so important? Because IRON BINDS OXYGEN!
The partnership between Iron and oxygen began over 3 billion years ago when cyanobacteria first introduced oxygen into earth’s water and atmosphere . As life continued to evolve, both iron and oxygen became indispensable for survival. Life can be terminated upon the failure of this partnership. Virtually all living cells are directly or indirectly affected by iron metabolism .
In the human body iron exists mostly in erythrocytes (red blood cells). The total body iron content in adult humans is approximately 50 mg/kg in men and 40 mg/kg in women .
Erythrocytes are able to deliver oxygen throughout the body because it contains hemoglobin [9, 10]. In vertebrates, hemoglobin contains four heme proteins. One iron ion is bound within each heme group, and each iron ion can bind one oxygen molecule. Therefore, a functional hemoglobin carries four iron ions and four oxygen molecules. Heme is the protein that carries BOTH iron AND oxygen .
The survival of eukaryotic organisms, including humans, depend upon an adequate supply of oxygen. Oxygen is consumed by mitochondria in the production of biochemical energy in the form of adenosine triphosphate (ATP).
Dr. Warburg observed tumor cells produced energy by fermenting glucose in glycolysis, leading to the belief that the irreversible damage of mitochondria as a result of fermentation processes was the cause of cancer. This belief associated the concept of damaged mitochondria with cancer relying mostly on glycolysis for energy production, dramatically lowering the requirement for oxygen as substrate to support mitochondrial OXPHOS. However, science has shown that cancer cells exhibit increased iron concentration. Would that not also increase oxygen levels as a result of elevated heme production?
It is now understood that hypoxic cancer cells, through the activation of HIF1a, utilize glucose as substrate to fuel glycolytic metabolism, releasing lactate as end product. Lactate is then converted into pyruvate which is used as substrate to fuel mitochondrial OXPHOS to produce energy (ATP) in oxygenated cancer cells .
In human non-small cell lung cancer, oxygen consumption, ATP generation, mitochondrial heme, heme synthesis and uptake, as well as oxygen-utilizing hemoproteins were ALL found to be significantly UPREGULATED, compared to non-tumorigenic cells . Why is heme so important to mitochondria?
Cancer cells upregulate iron levels because mitochondria oxidative phosphorylation (OXPHOS) is highly dependent upon heme. In fact, heme is so important to mitochondria that the eight-step biosynthesis of heme occur partly in mitochondria and cytoplasm.
Iron, Mitochondria & Cytochrome C
Most iron in mammalian systems is diverted to mitochondria so that a mitochondrial enzyme called ferrochelatase can complete the final step in heme synthesis by inserting a ferrous iron ion into protoporphyrin IX (a heme precursor). Ferrochelatase accepts only the ferrous form of iron. Yeast mitochondria in vitro, even when supplied with ample NADH as reducing agent, were unable to transport ferric iron for use by ferrochelatase in the synthesis of the porphyrin ring, Protoporphrin IX .
Only when supplied with ferrous iron is Protoporphyrin IX able to be integrated into heme proteins which are then incorporated into hemoglobin, dioxygenases and cytochromes .
Cytochrome C Heme Biogenesis in Mitochondria of Humans & Yeast
[Source: Robert G. Kranz,Cynthia Richard-Fogal,1 John-Stephen Taylor, and Elaine R. Frawley MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Sept. 2009, p. 510–528 Vol. 73, No. 3 1092-2172/09/$08.000 doi:10.1128/MMBR.00001-09]
In mitochondria, the last complex of the electron transport respiratory chain is Cytochrome c oxidase, or complex IV (CIV). 90% of cellular oxygen consumption is carried out by cytochrome c oxidase (CIV), and it is essential for ATP production in mitochondria .
ATP, Proton-Gradient & Ferrous Iron
The synthesis of ATP by the mitochondrial ATP synthase, which is responsible for the phosphorylation of adenosine diphosphate (ADP) to form ATP, is powered by a proton-motive gradient between the inner matrix of the mitochondria and the intermembrane space. This proton gradient is maintained by proteins in the electron transport chain and is energetically unfavorable, as protons need to be pumped from the matrix into the intermembrane space . To power the pumping of protons across the inner membrane into the intermembrane space, mitochondria use ferrous iron ions in the heme proteins of Cytochrome C.
The driving element of the proton-pumping process is actually dependent upon the net positive charge created by the oxidation of the ferrous iron ion in the heme enzyme in Cytochrome C. Proton pumping can only occur during the reductive phase, and only about one proton equivalent per fully reduced heme is pumped upon complete oxidation of the fully reduced heme enzyme by molecular oxygen . That also means if the iron in the heme is in the ferric (Fe2) state, proton-pumping cannot be completed. Why does heme only work with iron in the ferrous (Fe2+) reduced state?
To understand this primordial relationship, we need to understand how organisms used iron before the Great Oxygenation Event.
Cytochrome C, Iron & the Cyanobacteria: A Tale of Evolution
The earth was once a planet filled with acidic oceans rich in dissolved ferrous iron . A lack of oxygen did not prevent early Archean life forms from using the photon energy from the sun to perform life sustaining energy production through anoxygenic photosynthesis, where light is captured and converted to ATP WITHOUT the production of oxygen. Water, is therefore, not used as an electron donor. Instead, early Archean life forms used ferrous iron as an electron donor and its oxidation product, ferric iron, as an electron acceptor during anaerobic respiration .
The co-evolution of the cyanobacteria began to fill the earth’s atmosphere with oxygen. Cyanobacteria used the powerful energy from light photons to split water into oxygen, protons and electrons . The introduction of oxygen favored cells with the ability to transition between oxic-anoxic zones since oxygen was extremely toxic to Archean life. One of those life forms that successfully adapted was the cyanobacteria. Science now dates the origin of cyanobacteria to about 1 billion years BEFORE the Great Oxygenation Event 2.45 billion years ago . That would also mean that the cyanobacteria survived in an anaerobic environment with high ferrous iron .
The cyanobacteria as we know today, is an aquatic photoautotrophic prokaryote that contributes significantly to global photosynthesis. Cyanobacteria today, is still heavily dependent upon iron, easily exceeding requirements of other photosynthetic as well as non-photosynthetic prokaryotes . In fact, cyanobacteria thrive and bloom only in waters rich in ferrous (Fe2+) iron . To carry out photosynthesis, cyanobacteria requires iron. Light in the wavelengths of under 500 nm activates the uptake of ferrous iron during the light cycle of cyanobacteria . Why do cyanobacteria still require ferrous iron? Cyanobacteria have Cytochrome C, just like our mitochondria. Cytochrome c require reduced iron in heme to pump protons to generate energy .
Heme & the Janus-Faced Iron: The Vitamin C Connection
The history of heme is perhaps as old as the cyanobacteria. The use of heme in Cytochrome c probably existed for almost as long. All Cytochrome c are found outside of the mitochondria inner membrane, within the intermembrane space. Most people are familiar with the mitochondria cytochrome c that contain only a single heme group. In the vast world of prokaryotes, there are literally hundreds of different cytochrome c with structures and functions that are drastically different from mitochondria. Some of these cytochrome c contain more than TEN heme groups. But one thing remains constant among all these diverse cytochrome c. The heme groups always contain a ferrous iron ion. Cytochrome c must remain in the fully reduced form in order to be functional .
Iron deficiency has been found to dramatically reduce the expression of Cytochrome c in mitochondria . In fact, iron deprivation has been demonstrated to induce apoptosis via the collapse of mitochondrial membrane potential and release of cytochrome c from mitochondria into cytosol, initiating a chain of reactions that result in cell death .
Dramatic Reduction of Cytochrome c Proteins in Mitochondria of Mice Fed Iron Deficient Diet
[Source: Merrill et al. Nutrition & Metabolism 2012, 9:104 http://www.nutritionandmetabolism.com/content/9/1/104}
Why is iron in the reduced ferrous (Fe2+) form so important for life on earth?
The earth was originally a stew of reduced ferrous iron before the arrival of oxygen. Oxygen binds to heme in the Fe2+ ferrous form, and not the Fe3+ oxidized ferric form . All functional hemoglobin must have the reduced ferrous form of iron in heme groups order to transport oxygen .
Hemoglobin in red blood cells are active only when the iron in the heme is in the ferrous reduced state. In this state, the heme is able to bind oxygen reversibly. When the iron in heme is oxidized to the ferric state, the heme is inactivated, and the hemoglobin becomes a metalloprotein called methemoglobin (metHb). Methemoglobin cannot bind oxygen because the iron it carries is in the oxidized ferric form. This also means metHb is unable to deliver needed oxygen to cells and tissues .
Hemoglobin has four heme groups. One single oxidized heme will affect the other three units in the heme groups of the hemoglobin, shifting the oxygenation dissociation curve to the left towards higher affinities, hindering the release of oxygen to tissues .
The ability of iron to easily interconvert between ferrous (Fe2+) and ferric (Fe3+) states is a double-edged sword. The inclusion of Iron in iron-sulfur clusters allows efficient redox and electron transfer reactions for important proteins such as cytochromes, ferredoxins and dehydrogenases .
The price for this efficiency however, is the generation of reactive oxygen species (ROS) by free iron. Labile or free iron can be toxic as iron readily donate or accept electrons from neighboring molecules, causing damage through the Fenton/Haber-Weiss reactions.
The Haber-Weiss cycle is a two-step reaction where superoxide reduce ferric ions into ferrous ions. The reduced ferrous ions then react with hydrogen peroxide to form hydroxide ions and hydroxyl radicals, which then convert the ferrous ions back into the ferric form .
Free Heme & Ferric Iron Toxicity in Parkinson’s Disease
Hemoglobin in the ferric form has been shown to be highly unstable. In the ferric form, hemoglobin have been seen to lose heme at substantially higher rates than ferrous or ferryl forms . When hemoglobin lose heme, the resulting free heme known as hematin may be highly dangerous as they can cross cell membranes easily. If hematin movement is uncontrolled, the free heme and iron will add to the labile iron pool, producing reactive oxygen species via the Fenton reaction, causing massive oxidative stress and toxicity within cells that will result in lipid, protein, DNA damage and eventually cell death [33, 34].
Parkinson’s disease is characterized by the deposition of aggregated α-synuclein believed to be caused by reactive oxygen species emanating from mitochondrial dysfunction. A large portion of the toxic ROS products may actually be formed in the presence of ferric iron. Levin et al. demonstrated that the DIRECT interaction between α-synuclein oligomers with ferric Fe3+ iron but not ferrous Fe2+ iron produced distinct α-synuclein oligomers called Intermediate II oligomers .
Intermediate II oligomers is a toxic particle in vivo because they can form pores in biological membranes . The mechanism involved in pore formation has been associated with amyloid-related neurodegenerative disease due to its ability to increase membrane conductance that leads to neuronal cell death .
The most surprising finding by Levin et al. was that ascorbic acid, Vitamin C, inhibited the formation of ferric iron-dependent intermediate II oligomers in a dose-dependent manner. Vitamin C not only can dissolve Intermediate II oligomers, it is able to reduce Fe3+ to Fe2+. By lowering the available concentration of ferric iron, the formation of intermediate II oligomers were significantly reduced .
In physiological conditions, iron can exist both in the ferrous and ferric state. When there is oxygen, ferrous iron is spontaneously oxidized into the ferric oxidation state. The human body uses vitamin C to convert this fully oxidized ferric iron back into the reduced ferrous state. In fact proper iron absorption in the human body depends on vitamin C’s ability to convert ferric iron into ferrous iron.
Vitamin C, Ferrous Iron & HIF1a – A Tale of Plasma Membrane Redox Enzymes
A shocking study released in March 2018 by Scheers et al. revealed that of the three most common ‘bioavailable’ iron supplements – ferric citrate, ferric EDTA and ferrous sulfate, only the two ferric chelates increased cellular levels of tumor promoting onco-protein amphiregulin and its receptor EGFr. Ferrous sulfate had absolutely no effect on these tumor promoting proteins . Do our bodies harbor a preference for ferrous iron?
Supplementation with ferrous sulfate has been found to enhance hemoglobin levels in children, showing a more favorable result than supplementing with iron polysaccharide complex, which are in the ferric form . That is also the reason why humans absorb iron more readily when it is in the heme form. When we consume foods with iron from non-meat sources, or supplements in the ferric form, our bodies must convert the non-heme ferric iron into ferrous form before the iron can be fully absorbed .
The absorption of iron occur mostly in the duodenum and proximal jejunum in the small intestines. Iron cannot be absorbed unless it is in the ferrous Fe2+ form or bound by heme proteins. The conversion of the insoluble Fe3+ into the absorbable soluble Fe2+ is undertaken by a plasma membrane redox enzyme called duodenal cytochrome B (Dcytb).
Discovered only in 2006, duodenal cytochrome B (Dcytb) is a di-heme transmembrane protein that DEPENDS on ascorbate as an electron donor for the reduction of ferric iron into ferrous iron . Once ferric iron is reduced to ferrous iron in the intestinal lumen, it can be transported into cells. Both iron transporters and Dcytb can be upregulated by HIF under hypoxic environments .
Free heme with its ferric iron can cause cytotoxicity . Methemoglobin is a form of hemoglobin that has hematin instead of the regular ferrous heme. The iron in hematin is oxidized from the ferrous (Fe2+) to the ferric (Fe3+) state and is unable to transport oxygen because the heme in ferric form cannot bind oxygen. As a result, tissue hypoxia occurs when the concentration of this form of hemoglobin is elevated.
CYB5R, Cancer & Vitamin C- The HIF1a connection
Methemoglobinemia is a blood disease where excessive methemoglobin in blood cells cause decreased oxygen transport, leading to hypoxia and cyanosis (blue discoloration of peripherals). Methemoglobinemia is associated with defective functioning of a plasma membrane redox enzyme called CYB5R, cytochrome b5 reductase.
Cytochrome b5 reductase is a NADH dependent di-heme transmembrane protein that reduces methemoglobin from the inactive ferric form back into the active ferrous hemoglobin. The reduction of Fe3+ into Fe2+ also converts NADH into NAD+, highlighting the importance of plasma membrane REDOX enzymes outside of mitochondria .
Overexpression of CYB5R in mice resulted in the inhibition of chronic pro-inflammatory pathways, modest lifespan extension and reduced liver carcinogenesis in addition to improved mitochondria function and decreased oxidative damage .
There are two forms of CYB5R, a soluble form found in human erythrocytes, and a membrane bound form that is found in mitochondria, nucleus, endoplasmic reticulum and plasma membrane of ALL CELL TYPES. The interesting part about the membrane bound form of CYB5R is that this protein actively regenerates ascorbic acid on the outer mitochondrial membrane even though the reductase uses NADH as electron donor to complete redox cycling of iron ions [48. 49, 50]. Why does CYB5R actively regenerates ascorbic acid?
The treatment of choice for methemoglobinemia is methylene blue (MB). However, the use of MB may have serious complications, and is contraindicated in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Methylene blue can actually cause methemoglobinemia in high doses by its oxidant effect and induce hemolysis in cases of G6PD deficiency . Vitamin C, ascorbic acid, has been used successfully to treat methemoglobinemia instead of methylene blue. Case studies have shown the ability of ascorbic acid to normalize oxygen saturation within one hour of administration .
There is yet another important connection between CYB5R and Vitamin C that is most underappreciated. It has to do with the tumor suppressive effects of CYB5R. CYB5R is found to be expressed in ALL cancer cell types . The expression of CYB5R varies in different types of cancers. It is found to be abnormally inactivated in prostate and breast cancers and up-regulated in B cell acute lymphocytic leukemia [55,56,57].
One of the important anti-neoplastic effects of CYB5R is its ability to down-regulate the expression of VEGF vascular endothelial growth factor to inhibit angiogenesis, suppressing tumor formation. Hypoxia-induced factor alpha (HIF-1α) is the main activator of VEGF expression in tumorigenesis and wound healing. CYB5R can inhibit VEGF induced angiogenesis because it is able to suppress HIF1α . The inhibition of HIF1α is highly dependent upon the availability of ascorbic acid. That is probably one of the main reasons why CYB5R regenerate ascorbic acid. It requires adequate ascorbic acid to inhibit HIF1a.
Vitamin C, Iron & Cancer – The HIF1a Connection
HIF1a is now regarded as an effective prognostic marker of cancer aggressiveness because it is considered to be the master regulator of neoangiogenesis in cancer development. HIF1a is able to control the expression of a wide array of genes involved in cancer progression . Cancer cells are able to grow in a rapid, disorganized manner because they are able to promote the formation of new blood vessels by activating pathways such as HIF which regulates angiogenesis . Angiogenesis is deeply tied to iron homeostasis.
Iron deficiency has been shown to significantly promote VEGF by stabilizing HIF-1α . Cancer cells love iron because increased DNA synthesis demands support from additional iron uptake. Iron’s role in electron transfers make them indispensable in the maintenance of heme-containing enzymes . Both HIF1a and iron metabolism is upregulated in cancer because HIF1a has a prominent role in iron homeostasis. HIF is able to modulate iron metabolism by regulating the expression of iron-related proteins, including Dcytb which promote intestinal iron absorption, and iron transporters like DMT1 .
In order to exert gene modulating effects, HIF α-subunits must be stabilized for activation and interaction with HIF β-subunits. The effective activation/stabilization of HIF1a or degradation/inhibition of HIF1a, hinges upon the availability of oxygen, FERROUS IRON, and vitamin C, ascorbic acid. The specific molecular structure of ascorbic acid is most critical in the degradation process of HIF1a. …… To Be Continued ……
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