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Biological Energy - Charge Transfer Reactions in Mitochondrial Respiratory Chain

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    Biosynthesis, muscle contraction, cell movement, and heat production form the essential components of the life cycle. These mechanisms require energy which comes from – the main and at most – the only source of energy – sunlight. Sunlight is absorbed by pigments of plants and some bacteria during photosynthesis. When the plants are digested by animals the energy of synthesized chemical bonds is used in chemical reactions where some chemical bonds are disintegrated and new chemical bonds are formed. This is the way of energy transfer among biological systems in the living world.

    History

    At the end of 18th century Joseph Priestley, the discoverer of oxygen, showed in his experiments that plants produce oxygen and animals use it. Later, thanks to Antoine Lavoisier, it became known that animals absorb oxygen simultaneously with food “burning”. Now it is believed that production of oxygen is the result of plant photosynthesis and the absorption of oxygen by animals is the result of tissue respiration or, in other words, oxidation of nutrients.

    During the period of one year plants on land and in the ocean manipulate colossal amount of mass and energy. They digest 1.5∙1011 tons of carbon dioxide, 1.2∙1011 tons of water, produce 1011 tons of oxygen and store 6∙1020cal of the Sun energy in a form of chemical energy of photosynthesis products. Total amount of energy stored by plants during a year is approximately 1021 J which is 0.1% of the total flux of Sun energy which reaches the Earth (1024 J/year).

    The beginning of the bioenergetics is associated with the name of a German physician Julius Robert von Mayer who proclaimed one of the original statements of conservation and conversion of energy or the first law of thermodynamics: “energy can be neither created nor destroyed” (1841). He described the chemical process that is vital for all biological systems. Now we call this process oxidation.

    At the beginning of the 20th century Max Rubner showed that energy obtained by bacteria due to food digestion is divided into two parts. The first part is the energy that is secreted into the environment in the form of heat and the energy that is contained in the waste products. The second part is the energy which is stored up in the cell material. The sum of these two energies is equal to the internal energy of the taken food. Analogous experimental evidence of the first law of thermodynamics was obtained by Wilbur Olin Atwater when he studied thermal balance of a human with a calorimeter which was an isolated chamber where the human was placed.

    One of the primary achievements during the development of bioenergetics was the statement that all the energetic processes in living world are uniform either in a microorganism or in a human. The substances through which energy transfer is implemented are macroergic (high-energy) compounds that usually contain phosphate groups. In 1930s soviet biochemist Vladimir Aleksandrovich Engelgardt was the first who established the role of these compounds in the processes of energy conversion. Later, many investigators confirmed that the most important energy transformer and transducer of all these substances is adenosine triphosphate (ATP) which is oxidized while producing adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and transferring phosphate groups to other molecules.

    Since the discovery of oxidative phosphorylation by Engelgardt until 1970s a “chemical’ hypothesis was used to explain conjugation of oxidation and phosphorylation. The hypothesis assumed that energy released due to electron transfer during catabolism is conveyed to ATP molecule by means of intermediate high energy compounds. However, these compounds were not found. The updated hypothesis which was based on physical knowledge explained the phenomenon of oxidative phosphorylation. This idea is associated with the name of British biochemist Peter D. Mitchell. He studied processes happening in mitochondria in a small laboratory, created at his own expense, where he carried out his experiments on the ordinary pH-meter. By 1966 Mitchell published the first evidence of his chemiosmotic hypothesis that he claimed in 1961. Twelve years later his discovery was acknowledged and he was awarded the Nobel Prize for Chemistry in 1978.

    According to Mitchell, photosynthesis chain of electron transfer in chloroplast and chromatophores or respiration chain in mitochondria is inserted into membrane of these organelles and crosses it through. The electron transfer through the chains of enzymes is accompanied by the transport of positively charges hydrogen ions (protons) through the membrane. This causes the accumulation of electric potential difference on both sides of the membrane. In other words, electron transfer chains in membranes work as generators of electric current. This process would not be possible without the membrane that plays the role of an insulator with high electric resistance and separates the two groups of charges: negative charges on one side and positive charges on the other. Mitchell’s theory considers the membrane as one of the most important elements in conjugation processes.

    Principle of Energetic Conjugation

    In living systems there are many processes which result in reduced entropy and increased free energy. They are biosynthesis, active transport, phosphorylation of ATP, motion, transfer, etc. Mentioned processes are not thermodynamically favorable if we consider them apart from the other reactions that take place in a cell. However, energetic conjugation of these thermodynamically unfavorable reactions with favorable reactions allows unfavorable reactions to happen because favorable reactions provide energy for them. Energetic conjugation requires the two conditions to be satisfied:

    1. Free energy provided by “thermodynamically possible” reaction exceeds the energy consumed by “thermodynamically impossible” reaction. In other words, there should be access of energy considering its loss during energy transfer.
    2. The two conjugated reactions should have a common component. Those components include phosphates, electrochemical potential and other factors.

    As an illustration for the principle of energetic conjugation there could be a mechanical analog - motion of the two bound blocks in gravitational field (Figure 1).

    Figure \(\PageIndex{1}\): Motion of the two blocks in gravitational field. The blue block which is heavier goes down and its potential energy decreases releasing energy. The yellow block which is lighter uses this released energy and goes up with potential energy increase. Friction illustrates the loss of energy during its transfer.

    The biochemical example for the principle of energetic conjugation could be phosphorylation of glucose conjugated with ATP hydrolysis. Phosphorylation of glucose during glycolysis is an energy-dependent process. It is characterized by ΔG = + 13.4 kJ/mol (pH=7, T=37C˚) and cannot be executed spontaneously:

    \[ glucose + H_3PO_4 \rightarrow glucose-6-phosphate + H_2O \label{1}\]

    However, the process becomes possible when this reaction is conjugated with ATP hydrolysis. ATP hydrolysis itself proceeds according to the following scheme (2):

    \[ ATP + H_2O \rightarrow ADP + H_3PO_4 \label{2}\]

    This is an exergonic process where ΔG = – 30.5 kJ/mol (pH=7, T=37C˚). The conjugation of glucose phosphorylation and ATP hydrolysis is thermodynamically favorable ΔG = – 17.1 kJ/mol (pH=7, T=37C˚):

    \[ ATP + glucose \xrightarrow{Hexokinase} ADP + glucose-6-phosphate \label{3}\]

    Basic forms of energy storage in cells

    There are several basic forms of energy storage in the cells; each cell converts the energy of the surrounding sources (light, chemical energy) to one of the three forms of energy:

    1. Adenosine triphosphate (ATP)
    2. Electrochemical potential difference of a proton (\(Δµ_{H^+}\)) on the energy converting membranes of cells
    3. Electrochemical potential difference of sodium (\(Δµ_{Na^+}\)) on the energy converting membranes of cells

    If we use the analogy with finances we could say that a cell keeps a part of its capital in cash (soluble ATP) and a part of the capital on a bank account and often in two different banks (ΔµH+ and ΔµNa+). Sodium energetics relates more to a cell membrane and proton energetics is inherent to intracellular membranes and mitochondrial membranes, in particular.

    Charge transfer reactions in mitochondria

    Structure and function of mitochondria and their membranes

    Mitochondrion is an intracellular organelle approximately 2µm long and 1 µm wide. Usually, a cell contains around 2000 mitochondria which volume is 25% of that of the cell. Every mitochondrion is constrained by two membranes: smooth outer mitochondrial membrane and plicate inner mitochondrial membrane with protrusions called cristae (Figure \(\PageIndex{2}\)). Both membranes have the width of about 6 nm. They are separated from one another with the space of approximately 5-10 nm called intermembrane space.

    1024px-Mitochondrion_mini.svg (1).png
    Figure \(\PageIndex{2}\): Mitochondrion (CC0; Kelvinsong  via wikipedia).

    Mitochondrial membranes are permeable only for small uncharged molecules like О2, СО2, Н2О, NO, Н2О2. They block not only the products of intermediate metabolism (i.e. pyruvate and acetyl-CoA) but also inorganic ions (H+, Na+, K+, Ca+ Cl-). As a result of nonequilibrium distribution of ions mitochondrial matrix (Figure 2) is negatively charged relative to cytoplasm. Transmembrane electrical potential difference on mitochondrial membrane can be –200mV. This is much higher than the electrical potential difference on the cellular membrane (~ – 60mV).

    The main function of mitochondria is to capture substrates that are rich in energy (such as fatty acids, pyruvate, carbon backbone of amino acids) from cytoplasm and oxidize them with the formation of CO2 and H2O conjugated with ATP synthesis. The basic stages of this process are the formation of electrochemical gradient of protons and phosphorylation of ADP molecule. These processes occur on the inner mitochondrial membrane with the participation of mitochondrial respiratory chain and H+-ATP-synthase.

    Organization of mitochondrial respiratory chain

    Mitochondrial respiratory chain is a network of enzymes (complex I, complex III and complex IV) that catalyze the process of electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to oxygen. This process is conjugated with the generation of electrochemical gradient of protons.

    \[\ce{ NADH + H^{+} + 1/2 O2 \rightarrow NAD^{+} + H2O } \label{4}\]

    Due to the great difference between oxidative-reductive potentials of the acceptor (O2) and donor (NADH) of electrons the reaction (4) is highly exergonic. At standard conditions ΔG0’= – 19.2kJ/mol. In case the reaction could happen without the participation of enzymes, the great heat release would lead to the destruction of intracellular structures. However, reduced NADH is stable and is not oxidized by oxygen without enzymes. The enzymes that are part of respiratory chain break down the energy released in NADH and oxygen reaction in the way that it can be stored in the appropriate form for ATP synthesis.

    Respiratory chain includes three protein complexes (I, III and IV), that are incorporated into inner mitochondrial membrane, and two transferable molecules-carriers – ubiquinone (CoQ, QH, Coenzyme Q10, coenzyme Q) and cytochrome c (Figure 3). Succinate dehydrogenase that belong to citrate cycle can be considered as complex II of respiration chain. However, it transfers electrons from succinate to CoQ without transmembrane proton transfer. Sometimes, H+-ATP-synthase is considered as complex V although it does not participate in electron transfer.

    The complexes of mitochondrial respiratory system are built up of many peptides and contain a number of oxidative-reductive coenzymes connected to proteins. They are flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) (complexes I and II), Fe-S centers (clusters) (I, II and III) and heme groups (II, III and IV).

    The electron transfer from NADH to oxygen includes three stages which are catalyzed by the three lipoprotein complexes (I, II and IV). The complexes are transmembrane proteins that contact with water phases of either mitochondrial matrix or intermembrane space. Liquid lipid phase allows two-dimensional diffusion of complexes in the plane of membrane which provides the opportunity for the complexes to move closer to each other during transport processes.

    Figure 3 – Electron transport chain of mitochondria.

    Complex I

    NADH (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) catalyzes two energetically conjugated processes:

    1. Exergonic process of hydride-ion (2e-,1H+) transfer from NADH and proton to ubiquinone (CoQ, QH, Coenzyme Q10, coenzyme Q)
    2. Endergonic process of four protons transfer from matrix to intermembrane space.

    A proton transfers in the direction with electrochemical gradient that’s why it requires energy. Thus, complex I is a proton pump which works due to the energy of oxidative-reductive reactions. Active site of the complex I binds NADH. The two electrons released from NADH are transferred trough flavin mononucleotide (FMN) and Fe-S clusters to ubiquinone. Ubiquinone then binds the other active site which is located in the hydrophobic phase of the membrane. Then the following reaction takes place:

    \[ NADH + 5H^{+}_N + Q \rightarrow NAD^{+} + QH_2 + 4H^{+}_P \label{5}\]

    *Subscript index N (negative) indicates that protons belong to negatively charged matrix; subscript index P (positive) indicates that protons belong to positively charged intermembrane space of mitochondria.

    Energy is stored in the form of electrochemical gradient of protons.

    There are several other ways for electrons to be introduced into respiration chain.

    1. After succinate oxidation electrons are transferred to CoQ by complex II through bound FAD, Fe-S centers and heme of group b.
    2. Ubiquinone can receive electrons from acyl-CoA. The first stage of β-oxidation is catalyzed by acyl-CoA-dehydrogenase that transfers electrons from acyl-CoA through FAD to electron transferring protein flavoprotein (ETF).
    3. Electrons can also be introduced to respiration chain from cytoplasm and glycerol-3-phosphate, in particular. In this case FAD group of glycerol-3-phosphate dehydrogenase transfers electrons to ubiquinone that further is reduced to ubiquinol (QH2).

    Complex III

    Further, due to participation of complex III (coenzyme Q : cytochrome c — oxidoreductase, bc1 complex) electrons are transferred from ubiquinonol (QH2) to cytochrome c. Energy of ubiquinol oxidation is transformed by cytochrome c into energy of electrochemical gradient of protons due to the work of complex III. This process happens according to a scheme that is called Q-cycle (Figure 4).

    Figure 4 – Electron transfer through the complex III in inner mitochondrial membrane. Q-cycle.

    Semi reduced form of ubiquinone (∙QH) binds an electron received from complex I and a proton received from matrix and produces ubiquinol (Figure 4, stage 1). Next to the border of membrane with intermembrane space reduced ubiquinone (QH2) reacts with iron of heme bLand releases proton into water phase due to its oxidation. Ubiquinone itself turns into semi reduced form (∙QH) (semiquinone) (Figure 4, stage 2). Further the electron is transferred from heme bL to iron of heme bH which is closer to mitochondrial matrix. Then semiquinone (∙QH) gives the second electron to Fe-S centers. Meanwhile the second proton appears in the water phase of intermembrane space (Figure 4, stage 3). Further, the electron is transferred to the iron of cytochrome c1 and then to cytochrome c that oxidizes semiquinone (∙QH). Oxidized ubiquinone (Q) diffuses back to the side of the membrane which faces matrix. Here ubiquinone (Q) receives electron from bH and proton from water phase of the matrix (Figure 4, stage 4). As a result semiquinone (∙QH) accepts an electron from complex I and proton from matrix. Thus, ubiquinol (QH2) is formed. The Q-cycle can take place again. The overall chemical reaction occurring in complex III is as follows (6):

    \[ QH_2 + 2H^{+}_N + 2\:cyt\:c\:(Fe^{3+}) \rightarrow Q + 2\:cyt\:c\:(Fe^{2+}) + 4H^{+}_P \label{6}\]

    *Subscript index N (negative) indicates that protons belong to negatively charged matrix; subscript index P (positive) indicates that protons belong to positively charged intermembrane space of mitochondria.

    Complex IV

    Cytochrome c reduced after participation in Q-cycle transfers electrons to complex IV (cytochrome c oxidase). Complex IV delivers electrons through copper containing sites and hemes a and a3 to oxygen. This process is also conjugated with proton pumping from the matrix into intermembrane space (7):

    \[ 4\:cyt\:c\:(Fe^{2+}) + O_2 + 8H^{+}_N \rightarrow 4\:cyt\:c\:(Fe^{3+}) + 2H_2O + 4H^{+}_P \label{7}\]

    *Subscript index N (negative) indicates that protons belong to negatively charged matrix; subscript index P (positive) indicates that protons belong to positively charged intermembrane space of mitochondria.

    Further, the created proton potential on the inner mitochondrial membrane is converted into chemical energy stored in phosphate bonds of ATP molecules. This energy conversion is executed by H+-ATP-synthase that is the smallest of all known molecular motors. This enzyme can also work in the reverse direction, i.e. hydrolyze ATP if there is access of ATP molecules in mitochondrial matrix.

    According to the concepts of cell bioenergetics accepted today, cell respiration is executed on the basis of chemiosmotic transformations described in Peter D. Mitchell’s theory. It takes place on enclosed vesicles which are mitochondria where mitochondrial membrane serves as a container for respiratory chain enzymes, as a medium in which electrons are carried and as an insulator for protons that are on both sides of the lipid. During respiration energy of the electrons released from NADH is transformed into electric energy of proton gradient (ΔµH+) by protein complexes of mitochondrial respiratory chain. Then proton gradient ΔµH+ causes ADP phosphorylation that is ensured by H+-ATP-synthase. This enzyme transforms electrical energy of protons into energy of macroergic bonds of ATP. This is the basic idea of oxidative phosphorylation.

    References

    1. Grigory G. Martinovich, Bioenegretics and Metabolism (notes), Belarusian State University, 2012.
    2. Sergey N. Cherenkevich et al., Biological Membranes, Belarusian State University, 2009.
    3. Vladimir P. Skulachev et al., Principles of Bioenergetics, Moscow State University, 2010.
    4. Bruce Alberts et al., Molecular Biology of the Cell, 4th edition, New York: Garland Science, 2002.

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