MitoPedia: Substrates and metabolites

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MitoPedia: Substrates and metabolites

MitoPedia - high-resolution terminology - matching measurements at high-resolution.
The MitoPedia terminology is developed continuously in the spirit of Gentle Science.

»O2k-Publications: Substrate

ADPDAdenosine diphosphate is a nucleotid. In OXPHOS core metabolism, ADP is a substrate of ANT and ATP synthase in the phosphorylation system. ADP is the discharged or low-energy counterpart of ATP. ADP can accept chemical energy by regaining a phosphate group to become ATP, in substrate-level phosphorylation (in anaerobic catabolism), at the expense of solar energy (in photosynthetic cells) or chemiosmotic energy (respiration in heterotrophic cells). ADP is added to mitochondrial preparations at kinetically saturating concentrations to induce the active state for evaluation of OXPHOS capacity.
ATPTAdenosine triphosphate is a nucleotid and functions as the major carrier of chemical energy in the cells. As it transfers its energy to other molecules, it looses its terminal phosphate group and becomes adenosine diphosphate (ADP).
ActivityaThe activity (relative activity) is a dimensionless quantity related to the concentration or partial pressure of dissolved substances. The activity of a dissolved substance B equals the concentration, cB [mol·L-1], at high dilution divided by the unit concentration, c° = 1 mol·L-1:
aB = cB/c°

This simple relationship applies frequently to substances at high dilutions <10 mmol·L-1 (<10 mol·m-3). In general, the concentration of a solute has to be corrected for the activity coefficient (concentration basis), γB,

aB = γB·cB/c°

At high dilution, γB = 1.

For a dissolved gas G, the activity is the partial pressure, pG [Pa] (strictly: fugacity), divided by the unit partial pressure, p° = 1 Pa. The partial pressure is related to the concentration of the gas by the solubility, SG [Pa/mol] (see Oxygen solubility):

aG = cG·SG/p°

In general, the relative activity is defined by the chemical potential, µX

aX = exp[(µX-µ°)/RT]
Additive effect of convergent electron flowAα&βAdditivity describes the princple of substrate control of mitochondrial respiration with convergent electron flow. The additive effect of convergent electron flow is a consequence of electron flow converging at the Q-junction from respiratory Complexes I and II (NS or CI&II e-input). Further additivity may be observed by convergent electron flow through glycerophosphate dehydrogenase and electron-transferring flavoprotein complex. Convergent electron flow corresponds to the operation of the TCA cycle and mitochondrial substrate supply in vivo. Physiological substrate combinations supporting convergent NS e-input are required for reconstitution of intracellular TCA cycle function. Convergent electron flow simultaneously through Complexes I and II into the Q-junction supports higher OXPHOS-capacity and ET-capacity than separate electron flow through either CI or CII. The convergent NS effect may be completely or partially additive, suggesting that conventional bioenergetic protocols with mt-preparations have underestimated cellular OXPHOS-capacities, due to the gating effect through a single branch. Complete additivity is defined as the condition when the sum of separatly measured respiratory capacities, N + S, is identical to the capacity measured in the state with combined substrates, NS (CI&II). This condition of complete additivity, NS=N+S, would be obtained if electron channeling through supercomplex CI, CIII and CIV does not interact with the pool of redox intermediates in the pathway from CII to CIII and CIV, and if the capacity of the phosphorylation system (≈P) does not limit OXPHOS-capacity (excess E-P capacity factor is zero). In most cases, however, additivity is incomplete, NS < N+S.
Adenine nucleotidesANAdenine nucleotides, which are also sometimes referred to as adenosines or adenylates, are a group of organic molecules including AMP, ADP and ATP. These molecules present the major players of energy storage and transfer.
AerobicoxThe aerobic state of metabolism is defined by the presence of oxygen (air) and therefore the potential for oxidative reactions (ox) to proceed, particularly in oxidative phosphorylation (OXPHOS). Aerobic metabolism (with involvement of oxygen) is contrasted with anaerobic metabolism (without involvement of oxygen): Whereas anaerobic metabolism may proceed in the absence or presence of oxygen (anoxic or oxic conditions), aerobic metabolism is restricted to oxic conditions. Below the critical oxygen pressure, aerobic ATP production decreases.
Amount of substancen [mol]The amount of substance, n, is a base physical quantity, and the corresponding SI unit is the mole [mol]. Amount of substance (sometimes abbreviated as 'amount' or 'chemical amount') is proportional to the number of specified elementary entities, Ni of that substance i, and the universal proportionality constant is the reciprocal value of the Avogadro constant [1],
ni = Ni/NA

ni contained in a system can change due to internal and external transformations,

dni = dini + deni

In the absence of nuclear reactions, the amount of any atom is conserved, e.g., for carbon dinC = 0. This is different for chemical substances or ionic species which are produced or consumed during the advancement of a reaction, r,

Amount dn.png
A change in the amount of i, dni, in an open system is due to both the internal formation in chemical transformations, drni, and the external transfer, deni, across the system boundaries. dni is positive if i is formed as a product of the reaction within the system. deni is negative if i flows out of the system and appears as a product in the surroundings [2].
AnaerobicAnaerobic metabolism takes place without the use of molecular oxygen, in contrast to aerobic metabolism. The capacity for energy assimilation and growth under anoxic conditions is the ultimate criterion for facultative anaerobiosis. Anaerobic metabolism may proceed not only under anoxic conditions or states, but also under hyperoxic and normoxic conditions (aerobic glycolysis), and under hypoxic and microxic conditions below the limiting oxygen pressure.
AnaplerosisAnaplerosis is the process of formation of intermediates of the tricarboxylic acid cycle. Malic enzyme (mtME), phosphoenopyruvate carboxykinase (PEPCK), propionyl-CoA carboxylase, and pyruvate carboxylase play important roles in anaplerosis.
AnoxicanoxIdeally the term anoxic (anox, without oxygen) should be restricted to conditions where molecular oxygen is strictly absent. Practically, effective anoxia is obtained when a further decrease of experimental oxygen levels does not elicit any physiological or biochemical response. The practical definition, therefore, depends on (i) the techiques applied for oxygen removal and minimizing oxygen diffusion into the experimental system, (ii) the sensitivity and limit of detection of analytical methods of measuring oxygen (O2 concentration in the nM range), and (iii) the types of diagnostic tests applied to evaluate effects of trace amounts of oxygen on physiological and biochemical processes. The difficulties involved in defining an absolute limit between anoxic and microxic conditions are best illustrated by a logarithmic scale of oxygen pressure or oxygen concentration. In the anoxic state (State 5), any aerobic type of metabolism cannot take place, whereas anaerobic metabolism may proceed under oxic or anoxic conditions.
AscorbateAsIn respiratory assays for cytochrome c oxidase activity (Complex IV, CIV), ascorbate is added as regenerating system for maintaining TMPD in a reduced state. As has to be titrated into the respiration medium prior to the addition of TMPD, otherwise the reaction velocity of autoxidation is permanently elevated.
CarnitineCarCarnitine is an important factor for the transport of long-chain fatty acids bound to carnitine (carnitine acyltransferase) into the mitochondrial matrix for subsequent β-oxidation. There are two enantiomers: D- and L-carnitine. Only the L-isomer is physiologically active.
Cellular substratesCe; Cm1) Cellular substrates in vivo, endogenous; Ce. 2) Cellular substrates in vivo, with exogenous substrate supply from culture medium or serum; Cm.
Citrate synthaseCSCondensation of oxaloacetate with acetyl-CoA yields citrate as an entry into the TCA cycle, with CS located in the mt-matrix.
Coenzyme QQ, CoQCoenzyme Q, redox system (oxidized ubiquinone, partially reduced semiquinone, fully reduced ubiquinol) of the ET-pathway. More details » Q-junction.
Convergent electron flown.a.
Convergent electron flow
Convergent electron flow is built into the metabolic design of the Electron transfer-pathway. The glycolytic pathways are characterized by important divergent branchpoints: phosphoenolpyruvate (PEPCK) branchpoint to pyruvate or oxaloactetate; pyruvate branchpoint to (aerobic) acetyl-CoA or (anaerobic) lactate or alanine. The mitochondrial Electron transfer-pathway, in contrast, is characterized by convergent junctions: (1) the N-junction and F-junction in the mitochondrial matrix at ET-pathway level 4, with dehydrogenases (including the TCA cycle) and ß-oxidation generating NADH and FADH2 as substrates for Complex I and electron-transferring flavoprotein complex, respectively, and (2) the Q-junction with inner mt-membrane respiratory complexes at ET-pathway level 3, reducing the oxidized ubiquinone and partially reduced semiquinone to the fully reduced ubiquinol, feeding electrons into Complex III.
CreatineCrCreatine is a nitrogenous organic acid that occurs naturally in vertebrates and helps primarily muscle cells to supply energy by increasing the formation of adenosine triphosphate (ATP).
Cytochrome ccCytochrome c is a component of the Electron transfer-pathway (ET-pathway) in mitochondria. It is a small heme protein loosely associated with the outer side of the inner mitochondrial membrane. The heme group of cytochrome c transfers electrons from Complex III to Complex IV. The release of cytochrome c into the cytoplasm is associated with apoptosis.
Cytochrome c control factorFCFcThe cytochrome c control factor expresses the control of respiration by externally added cytochrome c, c, as a fractional change of flux from substrate state CHO to CHOc. In this flux control factor (FCFc), CHOc is the reference state with stimulated flux; CHO is the background state with CHO substrates, upon which c is added,
» MiPNet article
ET-pathway substrate typesn.a.See Electron transfer-pathway state
Electron transfer-pathway stateET-pathway state
SUIT-catg FNSGpCIV.jpg

Electron transfer-pathway states are obtained in mitochondrial preparations (isolated mitochondria, permeabilized cells, permeabilized tissues, tissue homogenate) by depletion of endogenous substrates and addition to the mitochondrial respiration medium of fuel substrates (CHNO) activating specific mitochondrial pathways, and possibly inhibitors of specific pathways. Mitochondrial electron transfer-pathway states have to be defined complementary to mitochondrial coupling control states. Coupling control states require ET-pathway competent states, including oxygen supply. Categories of SUIT protocols are defined according to mitochondrial ET-pathway states.

» MiPNet article
FADH2FADH2FAD and FADH2: see Flavin adenine dinucleotide.
Fatty acidFA

Fatty acids are carboxylic acids with a carbon aliphatic chain. The fatty acids can be divided by the length of this chain, being considered as short-chain (4–8 carbons), medium-chain (6–12 carbons) and long-chain (14-22 carbons) fatty acids.

Long-chain fatty acids must be bound to carnitine to enter the mitochondrial matrix, in a reaction that can be catalysed by carnitine acyltransferase. For this reason, long-chain fatty acids, such as palmitate (16 carbons) is frequently supplied to mt-preparations in the activated form of palmitoylcarnitine. Fatty acids with shorter chains, as octanoate (8 carbons) may enter the mitochondrial matrix, however, in HRR they are more frequently supplied also in the activated form, such as octanoylcarnitine.

Once in the mitochondrial matrix, the fatty acid oxidation (FAO) occurs, generating acetyl-CoA, NADH and FADH2. In the fatty acid oxidation pathway control state electrons are fed into the F-junction involving the electron transferring flavoprotein (CETF). FAO cannot proceed without a substrate combination of fatty acids & malate, and inhibition of CI blocks FAO. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
Fatty acid oxidationFAOFatty acid oxidation (β-oxidation) is a multi-step process by which fatty acids are broken down to generate acetyl-CoA, NADH and FADH2 for further energy production. Fatty acids (short chain with 4–8, medium-chain with 6–12, long chain with 14-22 carbon atoms) are activated by fatty acyl-CoA synthases (thiokinases) in the cytosol. The outer mt-membrane enzyme carnitine palmitoyltransferase I (CPT 1) generates an acyl-carnitine intermediate for transport into the mt-matrix. Octanoate, but not palmitate, (eight- and 16-carbon saturated fatty acids) may pass the mt-membranes, but both are frequently supplied to mt-preparations in the activated form of octanoylcarnitine or palmitoylcarnitine. Electron-transferring flavoprotein complex (CETF) is located on the matrix face of the inner mt-membrane, and supplies electrons from fatty acid β-oxidation (FAO) to CoQ. FAO cannot proceed without a substrate combination of fatty acids & malate, and inhibition of CI blocks FAO completely. Fatty acids are split stepwise into two carbon fragments forming acetyl-CoA, which enters the TCA cycle by condensation with oxaloacetate (CS reaction). Therefore, FAO implies simultaneous electron transfer into the Q-junction through CETF and CI.
Flavin adenine dinucleotideFAD, FADH2Flavin adenine dinucleotide, FAD and FADH2, is an oxidation-reduction coenzyme (redox cofactor; compare NADH). FMN and FAD are the prosthetic groups of flavoproteins (flavin dehydrogenases). Type F substrates (fatty acids) generate FADH2, the substrate of electron transferring flavoprotein (CETF). Thus FADH2 forms a junction or funnel of electron transfer to CETF, the F-junction (compare N-junction, Q-junction), in the F-pathway control state. In contrast, FADH2 is not the substrate but the product of succinate dehydrogenase (CII). FAD is the oxidized (quinone) form, which is reduced to FADH2 (hydroquinone form) by accepting two electrons and two protons.
Free radicalsA free radical is any atom or molecule that contains one or more unpaired electrons in an orbital. The degree of chemical reactivity depends on the localization of unpaired electrons. Free radicals are extremely reactive, and they can either donate or accept an electron from other molecules. Free radicals that include oxygen radicals and derivatives of oxygen are reactive oxygen species (ROS). Likewise, reactive nitrogen species (RNS) are nitric oxide-derived compounds. ROS/RNS include oxygen/nitrogen free radicals and non-radicals that are easily converted into radicals. Mitochondria are a main endogenous source of free radicals in cells and consequently are exposed to oxidative-nitrosative damage. Electron transfer in the electron transfer-pathway (ET-pathway) is not perfect, leading an electron leakage. This electron leakage permits the formation of ROS such as superoxide anion (O2•−), hydrogen peroxide (H2O2) and the hydroxyl radical (HO•).
GlucoseGlcGlucose, also known as D-glucose or dextrose, is a monosaccharide and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate.
Glutamic acid
Glutamic acid, C5H9NO4, is an amino acid which occurs under physiological conditions mainly as the anion glutamate-, G, with pKa1 = 2.1, pKa2 = 4.07 and pKa3 = 9.47. Glutamate&malate is a substrate combination supporting an N-linked pathway control state, when glutamate is transported into the mt-matrix via the glutamate-aspartate carrier and reacts with oxaloacetate in the transaminase reaction to form aspartate and oxoglutarate. Glutamate as the sole substrate is transported by the electroneutral glutamate-/OH- exchanger, and is oxidized in the mitochondrial matrix by glutamate dehydrogenase to α-ketoglutarate ( 2-oxoglutarate), representing the glutamate anaplerotic pathway control state. Ammonia (the byproduct of the reaction) passes freely through the mitochondrial membrane.
Glutamate-aspartate carrierThe glutamate-aspartate carrier catalyzes the electrogenic antiport of glutamate- +H+ for aspartate-. It is an important component of the malate-aspartate shuttle in many mitochondria. Due to the symport of glutamate- + +H+, the glutamate-aspartate antiport is not electroneutal and may be impaired by uncoupling. Aminooxyacetate is an inhibitor of the glutamate-aspartate carrier.
GlycerophosphateGpGlycerophosphate (synonym: α-glycerophosphate; glycerol-3-phosphate; C3H9O6P) is an organophosphate and it is a component of glycerophospholipids. The mitochondrial Glycerophosphate dehydrogenase complex oxidizes glycerophosphate to dihydroxyacetone phosphate and feeds electrons directly to ubiquinone.
Hydrogen peroxideH2O2
Hydrogen peroxide
Hydrogen peroxide, H2O2 or dihydrogen dioxide, is one of several reactive oxygen intermediates generally referred to as reactive oxygen species (ROS). It is formed in various enzyme-catalyzed reactions (e.g., superoxide dismutase) with the potential to damage cellular molecules and structures. H2O2 is dismutated by catalase to water and oxygen. H2O2 is produced as a signaling molecule in aerobic metabolism and passes membranes more easily compared to other ROS.
Hydrogen sulfideH2SHydrogen sulfide (H2S) is involved in signaling and may have have further biological importance.
Inorganic phosphatePiInorgnic phosphate (Pi) is a salt of phosphoric acid. In solution near physiological pH, the species HPO42- and H2PO4- dominate. See also: Phosphate carrier (Pic).
JmaxJmaxJmax is the maximum pathway flux (e.g. oxygen flux) obtained at saturating substrate concentration. Jmax is a function of metabolic state. In hyperbolic ADP or oxygen kinetics, Jmax is calculated by extrapolation of the hyperbolic function, with good agreement between the calculated and directly measured fluxes, when substrate levels are >20 times the c50 or p50.
Malic acid

Malic acid, C4H6O5, occurs under physiological conditions as the anion malate2-, M, with pKa1 = 3.40 and pKa2 = 5.20. L-Malate is formed from fumarate in the TCA cycle in the mitochondrial matrix, where it is the substrate of malate dehydrogenase oxidized to oxaloacetate. Malate is also formed in the cytosol. It cannot permeate through the lipid bilayer of membranes and hence requires a carrier (dicarboxylate carrier, tricarboxylate carrier and 2-oxoglutarate carrier). Malate alone cannot support respiration of mt-preparations from most tissues, since oxaloacetate accumulates in the absence of pyruvate or glutamate.

Malate is a type N substrate (N) required for the FAO-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) FAO-pathway could be overestimated due to a contribution of NADH-linked respiration, F(N) (see SUIT-002).
Metabolic control variableXA metabolic control variable, X, causes the transition between a background state, YX, and a reference state, ZX. X may be a stimulator or activator of flux, inducing the step change from background to reference steady state (Y to Z). Alternatively, X may be an inhibitor of flux, absent in the reference state but present in the background state (step change from Z to Y).
Methylmalonic acidMmaMethylmalonic acid (Mma) is a common intermediate in many catabolic processes. In methylmalonic acidemia mitochondrial dysfunction can be observed, related to accumulation of Mma and associated with neurological symptoms.
NADHNADHNAD+ and NADH: see Nicotinamide adenine dinucleotide.
NS-pathway control stateNS, CI&II
NS-pathway control
NS-pathway control is exerted in the NS-linked substrate state (flux in the NS-linked substrate state, NS; or Complex I&II, CI&II-linked substrate state). NS is induced in mt-preparations by addition of NADH-generating substrates (N-pathway control state, or CI-linked pathway control) in combination with succinate (S-pathway control state; S- or CII-linked). Whereas NS expresses substrate control in terms of substrate types (N and S), CI&II defines the same concept in terms of the convergent pathway to the Q-junction (pathway control). NS is the abbreviation for the combination of N- or NADH-linked substrates (CI-linked) and S- or succinate-linked substrates (CII-linked). This physiological substrate combination is required for partial reconstitution of TCA cycle function and convergent electron-input into the Q-junction, to compensate for metabolite depletion into the incubation medium. NS in combination exerts an additive effect of convergent electron flow in most types of mitochondria.
Nicotinamide adenine dinucleotideNADHNicotinamide adenine dinucleotide, NAD+ and NADH (pyridine nucleotide coenzymes, NAD and NADP), is an oxidation-reduction coenzyme (redox cofactor; compare FADH2). In the NADH Electron transfer-pathway state fuelled by type N substrates, mt-matrix dehydrogenases generate NADH, the substrate of Complex I (CI). The mt-NADH pool integrates the activity of the TCA cycle and various matrix dehydrogenases upstream of CI, and thus forms a junction or funnel of electron transfer to CI, the N-junction (compare F-junction, Q-junction). NAD+ and NADH are not permeable through the inner mt-membrane. Cytosolic NADH is effectively made available for mitochondrial respiration through the malate-aspartate shuttle or glycerophosphate dehydrogenase Complex.
OctanoateOcaOctanoate (octanoic acid). C8H16O2 Common name: Caprylic acid.
OctanoylcarnitineOctOctanoylcarnitine is a medium-chain fatty acid (octanoic acid: eight-carbon saturated fatty acid) covalently linked to carnitine, frequently applied as a substrate for fatty acid oxidation (FAO) in mitochondrial preparations.
Oxaloacetic acid
Oxaloacetic acid, C4H4O5, occurs under physiological conditions as the anion oxaloacetate2-, Oa. Oxaloacetate is formed from malate by MDH. Oa reacts with acetyl-CoA through citrate synthase to form citrate, or with glutamate through transaminase to form oxoglutarate and aspartate. Oa transport is restricted across the inner mt-membrane of various tissues. Oa is a potent inhibitor of succinate dehydrogenase.
2-Oxoglutaric acid
2-Oxoglutaric acid or alpha-ketoglutaric acid, C5H6O5, occurs under physiological conditions as the anion 2-Oxoglutarate2-, Og. 2-Oxoglutarate (alpha-ketoglutarate) is formed from isocitrate as a product of isocitrate dehydrogenase (IDH) in the TCA cycle, and is a substrate of oxoglutarate dehydrogenase (OgDH). The 2-oxoglutarate carrier exchanges malate2- for 2-oxoglutarate2- as part of the malate-aspartate shuttle. In the cytosol, oxoglutarate+aspartate are transaminated to form oxaloacetate+glutamate. Cytosolic malate dehydrogenase converts oxaloacetate+NADH to malate.
Molecular oxygen, O2 or dioxygen, has two atoms of oxygen, O, which is the chemical element with atomic number 8. The relative molecular mass of O2, Mr,O2, is 32 (or 31.9988). The element O has 8 protons, 8 neutrons and 8 electrons. In the figure, the two electrons in the first electron shell are not shown. Of the six electrons in the outer shell (blue bullets), one electron from each of the two atoms is shared in O2 forming the covalent bond, and one electron in each atom is unpaired.
P50p50p50 is the oxygen partial pressure at which (a) respiratory flux is 50% of maximum oxygen flux, Jmax, at saturating oxygen levels. The oxygen affinity is indirectly proportional to the p50. The p50 depends on metabolic state and rate. (b) p50 is the oxygen partial pressure at which oxygen binding (on myoglobin, haemoglobin) is 50%, or desaturation is 50%.
PalmitatePaaPalmitate is a term for the salts and esters of palmitic acid (CH3(CH2)14COOH). Palmitic acid is the first fatty acid produced during fatty acid synthesis and the precursor to longer fatty acids. Palmitate negatively feeds back on acetyl-CoA carboxylase (ACC), which is responsible for converting acetyl-CoA to malonyl-CoA, which in turn is used to add to the growing acyl chain, thus preventing further palmitate generation. In order to dissolve the water-insoluble sodium palmitate, BSA is needed to form the water-soluble compound called palmitate:BSA.
Palmitoyl-CoAPalmitoyl-CoA is a coenzyme A derivative of palmitate formed by acyl-CoA synthase. In contrast to medium- and short-chain acyl-CoA, palmitoyl-CoA cannot freely diffuse into the mitochondrial matrix. Formation of palmitoylcarnitine by CPTI is necessary prior to transfer into mitochondria for further fatty acid oxidation (β-oxidation).
PalmitoylcarnitinePalPalmitoylcarnitine is an ester derivative of carnitine (long-chain acylcarnitine) involved in the metabolism of fatty acids. Within the cell, palmitoylcarnitine is transported into the mitochondria to deliver palmitate for fatty acid oxidation and energy production.
PhosphatePiSee: Inorganic phosphate
PhosphocreatinePCrPhosphocreatine is a high energy compound in the skeletal muscle of vertebrates and is present in 4 to 5 times the concentration of ATP.
ProtonH+Proton and hydrogen ion, H+, are terms used synonymously in chemistry. A proton or hydrogen ion has no electrons and corresponds to a bare nucleus. The proton is a bare charge with only about 1/64,000 of the radius of a hydrogen atom, and so is extremely reactive chemically. The free proton has an extremely short lifetime in aqueous solutions where it forms the hydronium ion, H3O+, which in turn is further solvated by water molecules in clusters such as H5O2+ and H9O4+.

The transfer of H+ in an acid–base reaction is referred to as proton transfer. The acid is the proton donor and the base is the proton acceptor.

In particle physics, a proton is a subatomic particle with a positive electric charge. Protons and neutrons are collectively referred to as nucleons.
Pyruvic acid
Pyruvic acid, C3H4O3, is an alpha-keto monocarboxylic acid which occurs under physiological conditions mainly as the anion pyruvate-, P, with pKa = 2.5. Pyruvate is formed in glycolysis from phosphoenolpyruvate. In the cytosol, pyruvate is a substrate of lactate dehydrogenase. Pyruvate enters the mitochondrial matrix via a specific low Km' H+/monocarboxylate cotransporter known as the pyruvate carrier. Similarly, the plasma membrane of many cell types has H+/monocarboxylate cotransporter activity and pyruvate can thus be added as a substrate to intact cells. In the mt-matrix the oxidative decarboxylation of pyruvate is catalyzed by pyruvate dehydrogenase and yields acetyl-CoA. Pyruvate competitively reverses the inhibition of cytochrome c oxidase by cyanide. Pyruvate is an antioxidant reacting with hydrogen peroxide.
Reactive nitrogen speciesRNSReactive nitrogen species, RNS, are nitric oxide-derived oxidants. The main source of RNS is nitric oxide (NO•). NO• plays an important role in cell signaling and in oxidative-nitrosative stress.
Reactive oxygen speciesROSReactive oxygen species, ROS, are molecules derived from molecular oxygen, including free oxygen radicals, which are more reactive than O2. Physiologically and pathologically important ROS include superoxide, the hydroxyl radical and hydroxide ion, hydrogen peroxide and other peroxides. These are important in cell signalling, oxidative defence mechanisms and oxidative stress.
Substrate-uncoupler-inhibitor titrationSUITMitochondrial Substrate-uncoupler-inhibitor titration (SUIT) protocols are used with mitochondrial preparations to study respiratory control in a sequence of coupling and substrates states induced by multiple titrations within a single experimental assay.
Substrates as electron donorsMitochondrial respiration depends on a continuous flow of electron-supplying substrates across the mitochondrial membranes into the matrix space. Many substrates are strong anions that cannot permeate lipid membranes and hence require carriers.
Succinic acid
Succinic acid, C4H6O4, (butanedioic acid) is a dicarboxylic acid which occurs under physiological conditions as the anion succinate2-, S, with pKa1 = 4.2 and pKa2 = 5.6. Succinate is formed in the TCA cycle, and is a substrate of CII, reacting to fumarate and feeding electrons into the Q-junction. Succinate (CII-linked) and NADH (CI-linked) provide convergent electron entries into the Q-junction. Succinate is transported across the inner mt-membrane by the dicarboxylate carrier. The plasma membrane of many cell types is impermeable for succinate (but see Zhunussova 2015 Am J Cancer Res for an exception). Incubation of mt-preparations by succinate alone may lead to accumulation of oxaloacetate, which is a potent inhibitor of Complex II (compare Succinate and rotenone). High activities of mtMalic enzyme (mtME) prevent accumulation of oxaloacetate.
Succinate transportThe dicarboxylate carrier catalyses the electroneutral exchange of succinate2- for HPO4-2-.
Superoxide anion
Superoxide anion, O2•-, is a free radical formed in a one-electron reduction of molecular oxygen (red bullet in the figure), yielding a negatively charged molecule with a single unpaired electron (blue bullet on the left). It is highly reactive with organic compounds, and its intracellular concentration is kept under control by superoxide dismutase.
TMPDTmN,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride, TMPD, is applied as an artificial substrate for reducing cytochrome c in the respirometric assay for cytochrome c oxidase (CIV) activity. It is maintained in a reduced state by ascorbate and undergoes autoxidation as a function of oxygen pressure, TMPD, ascorbate and cytochrome c concentration.
TetrahydrofolateTHFTetrahydrofolate, THF, is the substrate in mitochondrial folate-mediated 1C metabolism, an NADH-linked pathway leading to the formation of formate which is exported to the cytosol.
Water, H2O, is widely used in the laboratory, particularly as a solvent and cleaning agent. Chemically pure water is prepared in various grades of purification: double distilled water (ddH2O) versus distilled water (dH2O or aqua destillata, a.d.) and deionized or demineralized water (diH2O) with various combination purification methods. When H2O is mentioned without further specification in published protocols, it is frequently assumed that the standards of each laboratory are applied as to the quality of purified water. Purification is not only to be controlled with respect to salt content and corresponding electrical conductivity (ultra-pure water: 5.5 μS/m due to H+ and OH- ions), but also in terms of microbial contamination.