Mitochondrial Function, ATP Production, Protein Folding & Molecular Hydrogen

Mitochondrial Function, ATP Production
Protein Folding & Molecular Hydrogen

Written by David Guez & Jim Wilson
30 August 2022

Foreword by Jim Wilson

Wow, what a mess the world has become regarding the biological strength of the human condition and the way our health systems are dealing with it. Inflammation and Oxidation are the obvious go-to observations that are treated and blamed for much of what we see. However, these are consequences after the primary causation, and we must look further back to find the root cause.

Homeostasis, balance and functioning at ease are what a biological organism or solution requires, and for it to be able to do that, it needs the appropriate resources to be available. If these resources are not available, then the biological organism will experience dis-ease and imbalance, which will ultimately progress to stress that the body will try to rectify, usually with a less efficient backup system. The key to evolution and survival as a species has a lot to do with the availability of these such backup systems. However, returning to homeostasis and the ability to function at ease is still required by the organism should long-term survival be assured.

When it comes to life on our planet, virtually all of it relies on an organelle called the mitochondria. This is the energy conversion device cells use to convert chemical energy to biological energy, which is called Adenosine TriPhosphate or ATP for short. Some cells contain only a few mitochondria to enable enough energy to be created for the necessary cellular process to function as intended, whereas other cells that require more energy may have thousands of them. Either way, if too little energy is produced in the cell, compensatory mechanisms are engaged, including prioritizing the available energy. This means that jobs do not get done or can be done poorly, which in turn means stress, failure and repair.

Herein lies the answers we seek when trying to address the many metabolic challenges we and other species face. This high-level paper attempts not only to identify how this energy conversion takes place inside the mitochondria but to identify and understand what this energy (ATP) does and what consequences may result from a shortfall or disruption of availability when challenges arise.

ATP

Adenosine triphosphate (ATP) is often described as the energy currency of life, and every cell needs to produce it to sustain its normal function. ATP is mainly formed into two cell compartments the cytoplasm periphery adjacent to the cell membrane and the mitochondria generally situated closer to the nucleus and the reticulum endoplasmic. While peripherical ATP production uses aerobic glycolysis and provides a fast response to rapid changes in ATP demand, the mitochondria provide a large amount of ATP, less sensitive to quick changes in demand. They can be described as satisfying the base-load demand¹. Interestingly manipulation of peripherical ATP demand, for example, by inhibition of Na+/K+ membrane pump, translates into a decrease in glycolysis, while manipulation of macromolecule synthesis translates into changes in respiration rate and thus changes in ATP by the mitochondria¹.

How ATP is Produced in the Mitochondria

The Respiration Chain

The mitochondrion is a cell organelle enclosed by a double membrane, an outer membrane and an inner membrane, separated by the intermembrane space. It is on the inner membrane surrounding the DNA-containing matrix that the respiratory complexes responsible for ATP production are localised. The respiratory chain comprises five protein complexes, Complex I, II, III, IV and V (Figure 1). The general purpose of the respiratory chain is to pump proton (H⁺) from the matrix to the intermembrane space resulting in a larger concentration of H⁺ in the intermembrane space than in the matrix, so doing creating a chemical and an electrical gradient. The creation of this gradient will provide the energy necessary for the synthesis of ATP by the ATP synthetase or complex V. The re-entry in the matrix of 3-4 moles of H⁺ translating into the production of one mole of ATP. The motrice force necessary to pump protons across the membrane is provided by electron transfer (Figure 1).

Complex I

Complex I is an NADH: ubiquinone oxidoreductase and one of the largest membrane-bound enzymes in the cell. Complex I contain 44 subunits in mammals², including 14 core subunits enabling its catalytic activities and conserved from bacteria to humans^3,4. Complex I catalyse NADH oxidation to NAD+ on the matrix side, capturing 2 electrons that are going to be transferred toward the membrane domain along a Flavin Mono Nucledotid (FMN) located near the site of NADH oxidation and a chain of iron-sulfur clusters up to the N2 cluster from which the electrons are going to be donated to the Ubiquinone-10 (Q10 or Q) in the Q chamber thus reducing the lipophilic Ubiquinone to Ubiquinol (QH2). QH2 will transport the electron across the inner membrane and donate them to complex III. The reduction of Ubiquinone to ubiquinol provides the energy necessary to transport four protons across the complex I membrane domain⁵. It is of note that NAD+ is reactivated to NADH via Beta oxidation of fatty acids and the tricarboxylic acid cycle taking place in the mitochondrial matrix.

Complex I is able to leak electrons at two sites⁶ and transfer them to Oxygen forming superoxide ions (O.^–) during the transfer of electrons from the NADH oxidase to the N2 cluster. The superoxide produced is found in the matrix, which can be converted in the matrix to hydrogen peroxide via a manganese superoxide dismutase (MnSOD). Complex I is the main source of ROS in the respiratory chain. If the production of superoxide is not balanced out by the activity of MnSOD, superoxide can deactivate complex I⁷.

Complex II

Complex II or succinate: ubiquinone oxidoreductase is composed of 4 subunits. The two largest subunits function as succinate dehydrogenase oxidising succinate to fumarate. The two electrons freed are transferred to a covalently bound FAD (Flavin adenine dinucleotide)⁸ reducing it to FADH2 from there the electrons are transferred to be ultimately donated to ubiquinone Q reducing it to ubiquinol. Complex II is not a major leakage site but may produce some superoxide on the matrix side of the intermembrane. Complex II does not pump H⁺.

Complex III

Complex III or ubiquinol cytochrome c reductase is a symmetrical dimer that contains 11 subunits each in mammals. Complex III receive successively two ubiquinol in the Q₀ chamber each time, transferring one electron to cytochrome c that will transfer them again to complex IV and one electron to its Q_i site where ubiquinone is reduced to ubiquinol in a process called the Q cycle. So one Q cycle sees the oxidation of two ubiquinol to ubiquinone at site Q₀, the reduction of cytochrome c and the reduction of one ubiquinone to ubiquinol Q_i. The transfer of electrons provides the energy necessary to transfer 4 H⁺ to the intermembrane space (two of which are pumped from the matrix). Complex III can produce superoxide at each Q site resulting in superoxide realise both in the intermembrane space where a copper-zinc superoxide dismutase (CuZnSOD) convert it to hydrogen peroxide. At the same time, the MnSOD in the matrix serves the same purpose⁶.

Complex IV

Complex IV or cytochrome c oxidase is composed in mammals of 13 subunits. It transfers electrons from cytochrome c to the terminal electron acceptor O₂ to generate H₂O, pumping four H⁺ from the matrix to the intermembrane space.

The ATP Synthase

The ATP Synthase (sometimes called Complex V) is a mitochondrial enzyme localized in the inner membrane adjacent to the respiratory complexes. It catalyzes the synthesis of ATP from ADP and phosphate using the energy derivated from the reentry flux of protons (H⁺) in the matrix from the intermembrane space. Thus the respiratory chain enabled the accumulation of H⁺ in the intermembrane space is the power that enables ATP synthesis in the mitochondria. While the energy needed to pump H⁺ from the mitochondrial matrix to the intermembrane space is derivated from the metabolism of nutrients that we consume, such as the tricarboxylic acid cycle (also called the Kreb cycle), the Beta oxidation of fatty acids or glutaminolysis.

The Action of Molecular Hydrogen on the Respiratory Chain

Very recently, it was demonstrated that H₂ supplementation suppressed superoxide production⁹ by complex I, its main producer. Furthermore, Ishihara et al. suggested that H₂ donated electrons in the Q chamber of complex I⁹. Two main mechanisms are possible, but given that Hydrogen evolution (the production of H₂ from two protons) by complex I in plants have recently been discovered¹⁰, the most likely is that complex I act as an oxygen insensitive hydrogenase capable of both using Hydrogen to reduce ubiquinone to ubiquinol or to accept electrons from ubiquinol and evolve Hydrogen gas from two protons. Regardless of how H₂ participate in the respiratory chain, it is demonstrated that H₂ supplementation translates into a more than 50% per min increase in ATP production by the mitochondria¹¹. An increase that appears to be at least partially uncoupled from nutrient intake. An increase in ATP production by the mitochondria following H₂ supplementation means that cells can divert the nutrient not used to produce energy to the production of the building block of the cells.

ATP - Beyond Energy

As we have seen previously, ATP is the main energy provider in biological systems. ATP, for example, provides the power necessary for muscle contraction, the functioning of neurones or the energy required for maintaining homeostasis. However, ATP fulfils non-energetic roles not less critical to the continuation of life. One of these fundamental roles is the capacity of ATP to increase the thermic stability of protein¹² and promote the correct folding of proteins¹³.

Proteins are chains of amino acids that are elongated by ribosomes in the endoplasmic reticulum (ER), based on the sequence of messenger ARN. Although the precise sequence of amino acids is essential to the protein function, its correct tri-dimensional structure is critical for the fulfilment of its biological function. The tri-dimensional structure is dependent on the appropriate protein folding. Protein folding occurs in the endoplasmic reticulum and is known to involve protein chaperones that facilitate the folding often using ATP as energy to promote a specific shape. However, it is only a small aspect of the folding process. Most importantly, ATP induces folding, inhibits aggregation and increases protein stability post folding^12,13.

The Folding Process

Protein translation and folding occur in the endoplasmic reticulum. First, as mentioned above, proteins are translated and elongated from messenger RNA. At this stage they do not yet possess their functional 3D structure. When immature chains expose segments that are prescript to be buried in the folded state, they are prone to accumulate and aggregate with other molecules via hydrophobic patches¹⁴. In other words, protein hydrophobic patches have a tendency to associate with each other to avoid contact with the solvent that is hydrophilic, forming dysfunctional protein aggregation that has been associated, for example, with Alzheimer’s disease and other neurodegenerative diseases. In order to promote correct protein folding and avoid aggregation, protein chaperones are present in the reticulum endoplasmic¹⁵, and capture unfolded protein or miss folded protein to enable their correct folding. Some of these chaperones use ATP as a source of energy¹⁴ to facilitate this process, others do not¹⁶. More recently, it was discovered that ATP is also able to facilitate the correct folding of proteins¹³. Despite all these control systems in place, misfolding does occur. Proteins that fail to fold properly are generally recycled. However, this is not always the case, and if a large number of misfolded proteins accumulate in the Reticulum endoplasmic, it triggers an ER stress response in the form of the Unfolded Protein Response.

The Unfolded Protein Response

The initial Unfolded Protein Response (UPR) is directed toward reestablishing homeostasis by increasing the expression of chaperone protein and boosting the production of ATP by the mitochondria¹⁷. It also enhances the degradation of slowly folding protein¹⁸, reducing the folding load of the ER, and increasing the degradation of unfolded proteins. The UPR can also boost the size of the ER to increase its folding capacity. On the other extreme, the UPR can lead to cell death by apoptosis¹⁸. But because misfolding can be caused by an infectious agent, ER stress also leads to the up-regulation of many pro-inflammatory cytokines, including TNFα, IL-1β, IFN-γ, IL-6, and IL-23¹⁹.

Folding and Autoimmune Disease

MHC molecules are categorized into class I and class II that are used by the immune system and normally present peptide fragments of protein to the immune system enabling the detection of foreign protein²⁰. Although MHC class I molecules are expressed in almost all cell types, MHC class II expression is restricted to certain cell types such as dendritic cells and B cells. Most non-immune cells normally do not express MHC class II molecules. However, in the case of humans, when cytokines such as interferon IFN-γ are produced, MHC class II expression is induced even on non-immune cells that do not express MHC class II molecules normally²⁰. During UPR, IFN-γ is expressed, enabling the expression of MHC class II molecule surprisingly, this molecule transports the misfolded protein in its entirety to the surface of the cell and presents it to the immune system enabling the development of antibodies toward this misfolded protein21 as it is the case in rheumatoid arthritis for example²². This work provides a direct link between the misfolding of proteins and the development of autoimmune diseases. In other words, it is possible that if misfolding were prevented or at least minimised, the autoimmune reaction would resolve itself. This is further reinforced by noting that ER stress always precedes the development of auto-immune diseases²³.

Folding and COVID-19

It is now well documented that Coronavirus replication causes ER stress and the Unfolded Protein Response^24–26. Furthermore, specific viral proteins have been shown to trigger the UPR, such as the spike protein, ORF2a, ORF6, ORF7a, ORF8ab, and ORF8b proteins^27–31. Interestingly pharmacological inhibition of the UPR was detrimental to virus replication³², suggesting that avoiding ER stress in the first place could be key to limiting virus replication. In this regard, it is interesting to note that the potential for increased ATP supply decrease with age³³ while Covid-19 susceptibility and/or severity increases with age^34,35. Given that the first event following the triggering of the UPR is an attempt to increase mitochondrial ATP supply to the ER in an attempt to re-establish homeostasis, improvement of mitochondrial ATP production could only be beneficial. This idea is further reinforced by the fact that some viral proteins are predicted to be targeted against the mitochondria, such as Non-Structural Protein 4 and 8 and ORF9a³⁶ and that Covid-19 infection results in a decreased mitochondrial ATP production³⁷ and a switch to aerobic glycolysis for the production of cellular ATP. Given that the main source of ER ATP during ER stress is mitochondrial, this will do little in receiving virus-related ER stress³⁸, thus contributing to increased cytokine production contributing negatively toward a cytokine storm. Thus increasing the basal production of mitochondrial ATP may be a good strategy to limit virus replication, reduce disease severity and avoid a highly detrimental cytokine storm.

Long COVID

Long Covid is driven by persistent inflammation of brain tissue in the absence of virus replication³⁹. This persistent inflammation is powered by the activation of microglia³⁹. This implies that microglia switch to glycolysis⁴⁰. Hydrogen supplementation has demonstrated its capacity to favour a reverse switch back to Oxidative Phosphorylation from glycolysis⁴¹. Given that glycolysis is necessary to the persistence of inflammation, it is expected that hydrogen supplementation will help resolve it.

ER stress and Protein misfolding have been identified in numerous diseases:

Firstly – in neurodegenerative diseases^42–44 such as:

Alzheimer disease.
Parkinson disease.
Spinocerebellar ataxia.
Amyotrophic lateral sclerosis.
Transmissible spongiform encephalopathies.
Multiple tauopathies.
Familial amyloidotic polyneuropathy.
Fronto temporal dementia.
Corticobasal degeneration.
Progressive supra nuclear palsy.
Dementia with Lewy bodies.

Second – Autoimmune Diseases^{20–22,45,46} such as:

Inflammatory myopathies.
Microscopic polyangiitis.
Rheumatoid arthritis.

And Third – during Coronavirus infections^24–26 such as:

SARS-CoV.
Murine hepatitis virus (MHV).
Alphacoronavirus transmisible gastroenteritis virus (TGEV).
Porcine epidemic diarrhea virus (PEDV).

Finally, Protein misfolding and ER stress are involved in both types of diabetes⁴⁷ and depression⁴⁸.

Autism (as an example)

In recent years some characteristics of the Autism Spectrum Disorder have emerged. First, Autism spectrum disorder is associated with neural inflammation (i.e. an elevated production of cytokines including IFN-γ), autoimmunity and autoantibodies^49,50. Second, autism spectrum disorder is associated with mitochondrial dysfunction⁵¹. Lastly, there is mounting evidence that Autism spectrum disorder is caused by protein misfolding^52–56. It is interesting to note that mitochondrial dysfunction can be at the same time the cause (not enough ATP production) and the consequence of protein misfolding following UPR (the stimulation of ATP production by the mitochondria can lead to an overproduction of reactive oxygen species, especially superoxide and trigger mitochondrial dysfunction. Furthermore, ER stress can trigger inflammation and the expression of MHC class II molecules²⁰ that will promote the autoimmune response.

Molecular Hydrogen

We know that molecular hydrogen supplementation increases the mitochondrial production of ATP by more than 50%¹¹ while suppressing the production of superoxide by the mitochondrial complex I⁹ (its main producer).

Molecular hydrogen supplementation has also demonstrated its anti-inflammatory potential by down-regulating NF- kb⁵⁷ (the transcription factor responsible for the production of cytokine following the Unfolded Protein Response (UPR).

An increased provision of ATP to the ER is predicted to facilitate correct protein folding by:

Providing the energy necessary for the correct functioning of the protein chaperone.
Promoting correct initial folding by itself¹³.
Protecting the ER from the aggregation of misfiled protein¹³, thus decreasing the risk of ER stress.
Preventing a run-away inflammatory response that increases the risk of developing an autoimmune response.

Thus the action of molecular hydrogen is predicted to significantly suppress the causes leading to ASD as shown in an animal model⁵⁸, including associated depressive boot since molecular hydrogen has proven potential in this area^59,60. Furthermore, Hydrogen supplementation could alleviate coronavirus-induced ER stress by increasing mitochondrial ATP production¹¹ as suggested by the observed improvement in COVID-19 patients during a recent clinical trial⁶¹. More generally, Hydrogen supplementation may be a path forward to alleviate ER stress and the UPR response in a wide variety of diseases and pre-diseases.

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