BIOCHEMISTRY (BIOENERGETICS)

ENERGY REQUIREMENTS FOR METABOLISM

• Metabolism encompasses the chemical processes used to synthesize complex molecules from basic precursor molecules (anabolism) and to break down complex molecules into less complex components (catabolism)

• Anabolism is the process by which living organisms build complex molecules from simpler ones. It is an endergonic process, meaning that it requires energy to occur. The energy required for anabolism is provided by the breakdown of complex molecules into simpler ones in a process called catabolism.

• Catabolism is the process by which living organisms break down complex molecules into simpler ones. It is an exergonic process, meaning that it releases energy. The energy released by catabolism is used to power anabolism and other cellular processes.

• The change in Gibbs free energy (ΔG) is a criterion used to predict whether or not a given chemical reaction will be spontaneous.

• Although it is impossible to measure the absolute value of free energy of a given molecule, it is possible to measure the change in total free energy, written ∆G, of a system (a collection of molecules at a given concentration, temperature, pressure, and pH) as a result of a particular reaction.

• The change in Gibbs free energy under standard conditions (ΔG°') is the change in Gibbs free energy when all components of a reaction are present at their standard states.

• An exergonic reaction is any reaction that releases Gibbs free energy, has a negative value for ∆G, and occurs spontaneously, an endergonic reaction is one that requires an input of Gibbs free energy, has a positive value for ∆G, and does not occur spontaneously.

• In a series of metabolic reactions, the overall Gibbs free energy change equals the sum of the Gibbs free energy changes for each individual reaction

a.      If the overall free energy change for a series of coupled reactions is negative (if ∆G3 < 0), the overall reaction is exergonic and occurs spontaneously in the forward direction

b.      If ∆G3 > 0, the overall reaction is endergonic and does not operate spontaneously in the forward direction

c.      If ∆G3 = 0, the reaction is at equilibrium and the rate of the forward reaction equals the rate of the reverse reaction

Sources of Free Energy

• The hydrolysis of adenosine triphosphate (ATP) provides the main source of free energy in biochemical reactions.

• The triphosphate group of ATP contains two high-energy phosphoanhydride bonds that, when hydrolyzed, each release approximately 7.3 kcal/mol of free energy; ∆G°’ for ATP hydrolysis to adenosine diphosphate (ADP) = -7.3 kcal/mol

• ATP is a free energy transmitter rather than a free energy reservoir. This means that ATP does not store a large amount of free energy, but instead it is constantly being recycled to transmit free energy from one reaction to another.

• The amount of ATP in a typical cell is usually only enough to meet free energy needs for a minute or two. This is because ATP is constantly being hydrolyzed to release energy for cellular processes. To meet ongoing needs for free energy, cells continually regenerate new ATP. This process is called ATP synthesis.

• Cells replenish ATP through three biochemical pathways: glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation

a.      In glycolysis, cells oxidize glucose to form pyruvate or (under anaerobic conditions) lactate; in the overall conversion of glucose to either pyruvate or lactate, each molecule of glucose provides enough energy to form two molecules of ATP

b.      In the TCA cycle (also called the Krebs cycle or the citric acid cycle), cells first convert pyruvate to acetyl coenzyme A (acetyl CoA) and then oxidize it completely to CO₂; the oxidation of two pyruvate molecules provides enough energy to form two molecules of the high-energy compound GTP, which can be converted to ATP

• Other nucleoside triphosphates (NTPs) such as guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP) also yield free energy through the hydrolysis of their phosphoanhydride bonds.

• Another high-energy molecule, acetyl CoA transfers acetyl groups in a way that is analogous to the transfer of phosphoryl groups by ATP

C. RELEASE OF ENERGY BY OXIDATION OF FOODSTUFFS

The human body obtains the energy needed for all its metabolic processes through the oxidation of carbohydrates, fats, and proteins in food.

a.  complex carbohydrate are catabolized to simple sugars, fats are catabolized to glycerol and fatty acids, and proteins are catabolized to amino acids.

b.  these simple sugars, fatty acids, and amino acids, are catabolized to a few common metabolic intermediates.

• Acetyl groups enter the  TCA cycle as acetyl CoA

• Electrons are transferred in the electron transport chain from NADH and FADH2 to special electron-carrier molecules.

• Oxidative Phosphorylation is the synthesis of ATP from ADP and Pi that occurs during the transport of electrons from NADH and FADH2 to oxygen.

D. REGULATION OF METABOLISM

• Prokaryotes regulate metabolism at the level of deoxyribonucleic acid (DNA) transcription; synthesis of the metabolic enzymes takes place in response to the cell's immediate needs.

• Eukaryotes use three primary routes for regulation of metabolism: enzyme control, differentiation of anabolic and catabolic pathways, and physical separation of metabolic pathways

a. Eukaryotes control the catalytic activity of enzymes by regulating the rate of enzyme synthesis and degradation through the use of allosteric inhibitors and activators and through covalent modification of the enzyme.

b. key regulatory steps; pathway differentiation permits separate but cooperative regulation of catabolism and anabolism and prevents energy waste caused by both paths operating simultaneously

c. anabolic and catabolic reactions generally are segregated in different parts of the cell

• The energy charge of a cell is the ratio of cellular ATP relative to AMP and ADP; cells maintain the energy charge within a narrow range; the balance of ATP versus AMP and ADP dictates whether ATP is required and must be synthesized or whether ATP is abundant (ready to be used)

Glycolysis

• Glycolysis is the first stage of cellular respiration and occurs in both aerobic (with oxygen) and anaerobic (without oxygen) conditions.

• Substrates: Glycolysis begins with one molecule of glucose (a six-carbon sugar) and results in the formation of two molecules of pyruvate (a three-carbon compound).

• Energy Investment and Payoff: Glycolysis consists of two main phases: the energy investment phase - two ATP molecules consumed and the energy payoff phase - four ATP molecules produced.

• NADH Production: glycolysis also generates NADH (nicotinamide adenine dinucleotide).

• Steps: Glycolysis comprises 11 enzymatic reactions, with each step catalyzed by a specific enzyme.

• End Products: The final products of glycolysis are two molecules of pyruvate, along with two ATP molecules and two NADH molecules per glucose molecule.

• Regulation: Glycolysis is tightly regulated to maintain cellular energy balance.

• Importance: Glycolysis is a central metabolic pathway and it serves as a critical energy source

• Location: Glycolysis takes place in the cytoplasm of the cell.

The Glycolytic pathway

• Glycolytic pathway consists of three (3) stages; Priming stage, Splitting stage, Oxidoreduction-phosporalation

Priming stage - steps 1 & 2

Splitting stage - steps 3, 4 & 5

Oxidoreduction-phosporalation stage - steps 6 - 11

Priming Stage

Step  1 - Glucose Phosphorylation:

   - Enzyme: Hexokinase (or Glucokinase in the liver)

   - Substrate: Glucose

   - Product: Glucose-6-phosphate

Step 2 - Isomerization:

   - Enzyme: Phosphoglucose Isomerase

   - Substrate: Glucose-6-phosphate

   - Product: Fructose-6-phosphate

Splitting Stage

Step 3 -  Second Phosphorylation:

   - Enzyme: Phosphofructokinase-1 (PFK-1)

   - Substrate: Fructose-6-phosphate

   - Product: Fructose-1,6-bisphosphate

Step 4 - Cleavage:

   - Enzyme: Aldolase

   - Substrate: Fructose-1,6-bisphosphate

   - Products:  DHAP and G3P

Step 5 - Isomerization (Interconversion):

   - Enzyme: Triosephosphate Isomerase

   - Substrate: Dihydroxyacetone phosphate (DHAP)

   - Product: Glyceraldehyde-3-phosphate (G3P)

Oxidoreduction-phosporalation Stage

Step 6 - Oxidation and ATP Generation:

   - Enzyme: Glyceraldehyde-3-phosphate Dehydrogenase

   - Substrate: Glyceraldehyde-3-phosphate (G3P)

   - Products: 1,3-bisphosphoglycerate and NADH

Step 7 - ATP Generation:

   - Enzyme: Phosphoglycerate Kinase

   - Substrate: 1,3-bisphosphoglycerate

   - Product: 3-phosphoglycerate

Step 8 - Substrate-Level Phosphorylation:

   - Enzyme: Phosphoglycerate Mutase

   - Substrate: 3-phosphoglycerate

   - Product: 2-phosphoglycerate

Step 9 - Dehydration:

   - Enzyme: Enolase

   - Substrate: 2-phosphoglycerate

   - Product: Phosphoenolpyruvate (PEP)

Step 10 - ATP Generation and Pyruvate Formation:

    - Enzyme: Pyruvate Kinase

    - Substrate: Phosphoenolpyruvate (PEP)

    - Product: Pyruvate

Step 11 - Final Pyruvate Formation:

    - Enzyme: Pyruvate Kinase

    - Substrate: Phosphoenolpyruvate (PEP)

    - Product: Pyruvate

III.  THE TRICARBOXYLIC ACID (TCA) CYCLE

A. GENERAL INFORMATION

1. The TCA cycle (also called the citric acid cycle or Krebs cycle) is a series of enzyme-catalyzed reactions occurring only in the presence of oxygen

• mitochondria of eukaryotic organisms 

• plasma membrane of prokaryotic organisms

Individual two-carbon units are carried into the TCA cycle as molecules of acetyl CoA and leave the cycle as CO2

2. At the end of the TCA cycle

• oxidized completely to six CO2 molecules

Through glycolysis, each glucose molecule is converted to two molecules of pyruvate Through oxidative decarboxylation, two pyruvate molecules yield two CO2 molecules Through the TCA cycle, two acetyl CoA molecules yield four CO2 molecules

a.      The enzyme pyruvate dehydrogenase catalyzes the oxidative decarboxylation of pyruvate to CO2 and acetyl CoA

• NADH (reduced form of NAD)

  -matrix of mitochondria

b.      Pyruvate dehydrogenase is a complex enzyme that contains three catalytic subunits

• pyruvate dehydrogenase

• dihydrolipoyl transacetylase

• dihydrolipoyl dehydrogenase

c.      In addition to its requirements for CoA and NAD, pyruvate dehydrogenase requires the cofactors

d.      The activity of pyruvate dehydrogenase is inhibited by elevated concentrations of NADH, acetyl CoA, and GTP (Guanosine triphosphate) and is activated by AMP (Adenosine monophosphate)

e.      In mammals, pyruvate dehydrogenase is inhibited via phosphorylation (covalent modification)

• Inactive

3.  The TCA cycle is the final common path for the oxidation of amino acids, fatty acids and carbohydrates.

a.      In the beginning of the cycle, acetyl CoA forms a complex with Oxaloacetate

b.      At the completion of the cycle, oxaloacetate is regenerated to complex with a new molecule of acetyl CoA

c.      In three different oxidation-reduction reactions of the cycle, NAD is reduced to NADH; in one oxidation-reduction reaction FAD reduced to FADH2

d.      The electrons caried by NADH and FADH2 ultimately enter the chain

4.  Because the the TCA pathway is cyclic, it has no ultimate products; TCA intermediates serve as building blocks for other biosynthetic reactions.

B. REACTIONS OF THE TCA CYCLE

STEP 1

Citrate Synthase catalyzes the condensation of acetyl CoA and Oxaloacetate to form Citrate; Citrate performs other roles besides energy generation in TCA cycle; its carbons are used as building blocks for such biosynthetic reactions as gluconeogenesis, and it is allosteric regulatory for other enzymes, such as phosphofructokinase, in glycolysis.

STEP 2

The enzyme Aconitase catalyzes the isomerization of Citrate to Isocitrate via the enzyme-bound intermediate, cis-aconitate.

STEP 3

Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of Isocitrate to Alpha Ketoglutarate and CO2; this reaction is the rate limiting step of the TCA cycle

STEP 4

The second oxidatively decarboxylation of the TCA cycle; Alpha ketoglutarate is oxidatively decarboxylated to CO2 and Succinate, which then complexes with the CoA to yield Succinyl CoA

STEP 5

The high-energy thioester bond of Succinyl CoA is hydrolyzed to form Succinate and CoA; in mammals, the energy released from hydrolysis of the thioester bond is used for substrate level phosphorylation of GDP to GTP; Succinyl CoA synthetase catalyzes this reaction.

STEP 6

Succinate Dehydrogenase catalyzes the oxidation of Succinate to Fumarate.

STEP 7

The enzyme Fumarase catalyzes the hydration of the Fumarate double bond to form Malate.

STEP 8

Malate Dehydrogenase catalyzes the oxidation of malate to Oxaloacetate, completing the cycle; NAD+, the required cofactor, is reduced yielding the third and last NADH + H of the TCA cycle

In terms of Stored chemical energy, one glucose molecule entering the TCA cycle as two molecules of acetyl CoA yields two molecules of GTP, six molecules of NADH + H and two molecules of FADH2.

C. GLYOXYLATE CYCLE

The Glyoxylate cycle, which resembles the TCA cycle, enables plants and bacteria to use acetate or other compounds that can be converted to acetyl CoA to generate energy and provide biosynthetic products.

DIFFERENCE BETWEEN GLYOXYLATE CYCLE AND TCA CYCLE

• Two acetyl units enter the Glyoxylate cycle together; acetyl units enter the TCA cycle one at a time.

• One acetyl unit forms Isocitrate, just as in the normal TCA cycle; however rather than forming a-ketoglutarate and subsequently Succinyl CoA, this Isocitrate molecule is cleaved yield Succinate, the product of the cycle, and Glyoxylate

• Glyoxylate reacts with the second molecule of Acetyl CoA to form malate, which is then oxidized to Oxaloacetate

• Plants and bacteria use the newly formed Oxaloacetate to make glucose through a process called Gluconeogenesis

IV. OXIDATIVE PHOSPHORYLATION IN THE RESPIRATORY CHAIN

A. GENERAL INFORMATION

• Oxidative phosphorylation is a process whereby ATP is produced as electrons are shuttled through the energy-generating components of the respiratory chain to molecular oxygen, forming water

  A. Electrons are donated from molecules of NADH and FADH2 produced in the complete oxidation of glucose to CO2 and water; NADH and FADH2 are oxidized in oxidative phosphorylation.

(1)   Two molecules of NADH result from glycolysis of one molecule of glucose

(2)   Two molecules of NADH result from the decarboxylation of two pyruvate molecules to acetyl CoA in the reaction linking glycolysis to the TCA Cycle

(3) six molecules of NADH and two molecule of FADH2 result from the TCA cycle oxidation of two acetyl CoA molecules

b.) The basic oxidative phosphorylation reaction is simply ADP + P-> ATP

c.) The oxidation-reduction reactions of oxidative phosphorylation are a separate biochemical event linked to the formation of ATP; the two reactions are linked by a Proton-motive force

d.) The result is that glucose is completely oxidized to CO2 by the end of the TCA cycle and water by the end of oxidative phosphorylation

• During oxidative phosphorylation, the oxidation of each NADH produces three molecules of ATP; the oxidation of each FADH2 produces two molecules of ATP

• Oxidative phosphorylation is the major source of ATP for aerobic organisms

• in eukaryotes, oxidative phosphorylation occurs in the mitochondrial inner membrane; in prokaryotes, it occurs in the cytoplasmic membrane.

B. Energetics of oxidative phosphorylation

• As electron are transferred sequantially from NADH and FADH2 to O2 (the final electron  acceptor) protons (H+) are translocated (”pumped”) out of the mitochondrial matrix into the intermembrane space between the inner mitochondria membrane and the outer mitochondrial membrane.

• Pumping of H+ from the mitochondrial matrix into the intermembrane space results in both a pH gradient and generation of an electrical potential across the inner mitochondrial membrane: this concentration gradient and electrical potential  tends to drive H+ back through the inner mitochondrial membrane and into the mitochondrial matrix.

• The movement of H+ back into the mitochondrial matrix is linked to an enzyme complex called ATP synthetase or ATPase: as the proton move through this enzyme complex, they foster the synthesis and release of ATP from ADP and PI

• Complete oxidation of one molecule of glucose (via glycolysis, the TCA cycle, and the respiratory chain) to CO2 and water yields either 38 or 36 molecules of ATP, depending on how the NADH produced in glycolysis in transported  into the motochondria to participate in the respiratory chain.

• Thirty-two of the 36 (or 34 of the 38) ATP molecules produced from the complete oxidation of one molecule of glucose (via glycolysis, the TCA cycle, and the repiratory chain) to CO2 and water are produced by oxidative phosphorylation. When reviewing the following sites of ATP production, recall that one molecule of glucose is spilt into two three-carbon units, doubling the number of NADH and FADH2 produced.

C. Respiratory Chain Components

• The respiratory chain transfer electrons and is composed of many different complexes, including proteins that acts as enzyme in electron transfer as well as specialized proteins called cytochromes; a cytochromes is an electron transferring protein with a heme prosthetic group.

• As electrons flow from NADH to O2, they are transferred sequentially to two intermediate compounds, coenzyme Q (CoQ) and cytochrome c (Cyt c) and then ultimately to molecular oxygen from Cyt c. 

Three different large enzyme complexes catalyze the flow of electrons between CoQ, Cyt c, and oxygen.

• NADH-CoQ reductase catalyzes electron flow between NADH and CoQ

• Cytochrome reductase catalyzes electron flow between CoQ and Cyt c

• Cytchorome oxidase catalyzes electron flow between Cyt c and O2 to form H2O; oxygen has a high affinity for electrons and is relatively unreactive, making it good choice for a terminal electron acceptor

The three enzyme complexes are the site of both H+ pumping and movement of electrons: the actual groups that carry electrons in the repiratory chain enzymes are flavin, iron, sulfur atoms proteins .

Electrons in the respiratory chain are first transferred from NADH to CoQ via the NADH-CoQ reductase complex, the first of the three enzymatic complexes that both transfer electrons and act as proton pumps.

a. The NADH-CoQ reductase complex first accepts pair of electrons from NADH at one of its prosthetic groups, flavin mononucleotide (FMN), reducing this prosthetic group to FMNH2: if only one electron is transferred, an unstable intermediate of CoQ, called a semiquinone (QH) is formed.

b. Electrons are then transferred from FMNH2 to a second prosthetic group of NADH-CoQ reductase, the iron sulfur protein also called nonheme iron proteins because the iron atom is bound to sulfur instead of heme.

c. Electrons are then transferred from the iron sulfur prosthetic group to the oxidized form of CoQ( also called ubiquinone); CoQ accepts electrons to form the reduced compound CoQH2 (also called ubiquino)

d. The second electron is transferred to heme groups in Cyt b: Cyt b donates electron this electron to QH, reducing to CoQH2: Cyt b is an electron recycling device that enables ubiquinol, a two electron carrier, to transfer one electron to a time to an iron sulfur cluster to cytochrome reductase, a single-electron carrier.

The third enzyme complex in the respiratory chain, the cytochrome oxidase complex, transfer electrons form Cyt c to O2 to  form H2O: four electrons are needed to comletely reduce one molecule of O2 to two molecules of H2O: this is the last of the three proton pumps

a. Cytochorome oxidase contains cytochrome with two different heme group (Cyt a and Cyt a3) and two copper ions (CuA and CuB)

b. Cyt c donates its electron to the Cyt a-CuA complex, then one electron at a time is transferred to the Cyt a3-CuB complex, then to O2, thus producing two H2O molecules.

The movement of electrons through all three enzymes complexes of the respiratory chain results in pumping of H+ from the matrix of the mitochondrial to the intermembrane space; the resulting difference in electrical potential and pH between matrix and the intermembrane space drives the production of ATP through a mechanism called the proton-motive force.

The NADH molecules produced in glycolysis  are the only glycolytic NADH molecules produced outside the mitochondria; to enter the respiratory chain, the NADH molecules must first traverse the mitochondrial membrane through two different shuttle mechanisms.

a.      Actually, neither shuttle mechanism transfer the NADH molecule across the inner mitochondrial membrane; rather, the shuttle transfer the electrons from NADH

b.      The two shuttle mechanism yield different amounts of ATP; the glycerol phosphate shuttle yields two molecules of ATP per NADH; the malate aspartate shuttle yields three molecules of ATP per NADH

c.       The glycerol-phosphate shuttle, active in muscle cells, begins when a dihydroxyacetone phosphate molecule in the cytosol is reduced to glycerol-3-phosphate by accepting electrons from cytosolic NADH that result from glycolysis.

(1)    The glycerol-3-phosphate molecule diffuses into the mitochondria delivers its electrons to FAD to form FADH2 and is simultaneously oxidized back to dihydroxyacetone phosphate

(2)    The recreated dihydroxyacetone phosphate then exits the mitochondria, thus completing the shuttle

(3)    FADH2 produced within the mitochondria enters the respiratory chain at CoQ, by passing the NADH-CoQ complex, which is site of a proton pump; thus, only two ATP molecules are produced

d. The malate-aspartate shuttle, active in heart and liver cells, functions because malate can cross mitochondrial membrane while oxaloacetate cannot; in the cytosol, an oxaloacetate molecule accepts electrons from NADH produced during glycolysis and is reduced to malate.

(1) The malate molecule crosses into the mitochondrion and in oxidized to oxaloacetate, oxaloacetate is transaminated to aspartate with the simultaneous production of NADH + H+ from NAD+

(2) Aspartate then leaves the mitochondria and diffuses into the cytosol, where it is converted back to oxaloacetate to complete the shuttle

(3) The NADH generated within the mitochondria enters the respiratory chain at NADH-CoQ reductase to produce three molecules of ATP

RESPIRATORY CHAIN

Generation of ATP by proton-motive force

The flow of electrons from NADH to 02 is a thermodynamically favorable reaction; the standard free energy change (∆Gᵒ’) for the reaction 1 ⁄2 02 + NADH + H+ + NAD can be calculated

A. For this reaction, which is the overall result of the electron transport of NADH electrons, ∆Gᵒ’ = - 52.6 kcal/mol

B. The free energy expended during the synthesis of one molecule of ATP from ADP is + 7.3 kcal/mol (∆Gᵒ’ = +7.3 kcal/mol)

C. The exergonic formation of water in the respiratory chain provides energy for the endergonic formation of ATP.

ATPase, located in the inner mitochondrial membrane, is composed of three different proteins subunits: F1 , F0 and a stalk protein

• F1 composed of five polypeptides and located on the matrix side of the inner mitochondrial membrane, catalyzes the reaction ADP + P1 ↔ ATP.

• F0m composed of four polypeptides and spanning the inner mitochondrial membrane, is the membrane channel through which H+ passes.

• The stalk protein links F0 to F1

• The ADP concentration is a major factor in determining the rate of oxidative phosphorylation, a low ADP concentration increases the rate of oxidative phosphorylation

• Electrons usually are transferred in the respiratory chain only when ATP is needed, this is called respiratory control.

The chemiosmotic hypothesis describes how the proton gradient between the mitochondrial matrix and the intermembrane space results in production of ATP.

a. In the respiratory chain, the transfer of electrons from one complex to the next leads to H+ pumping from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space.

b. H+ pumping results in a buildup H+ and a positive electrical potential on the outer side of the inner mitochondrial membrane.

c. The pH gradient and membrane electrical potential generate a protonmotive force

   (PMF), which does not actually form ATP, but rather allows ATP to be released from ATPase, the enzyme that catalyzes in synthesis.

d. The chemiosmotic theory does not describe how the movement of H+ through ATPase causes the generation of ATP, the theory merely describes how ATPase forms a channel for the redistribution of H+ while somehow simultaneously generating ATP.

e. A pH gradient is generated at each of the three electron transfer complexes where H+ pumping occurs (N ADH-CoQ reductase, cytochrome reductase, and cytochrome oxidase); one molecule of ATP is synthesized at each site of H+ pumping.

 f. The P:O ratio, the number of molecules of inorganic phosphate incorporated into ADP per atom of oxygen consumed, is an index of oxidative phosphorylation.

g. Because electrons from NADH are transferred through all three respiratory complexes at which H+ pumping can occur, the oxidation of NADH has a P:O ratio of 3; because electrons from FADH2 enter the respiratory chain at CoQ thereby passing NADH-Q reductase, the oxidation of FADH2 has a P:O ratio of 2.

Respiratory chain inhibitors and uncouplers

The three H+ pumping sites of the respiratory chain can be blocked by various inhibitors.

• NADH-CoO reductase is inhibited by rotenone (a fish protein) and amytal (a barbiturate); these compounds do not inhibit the oxidation of FADH2, because FADH2 enters the respiratory chain after this inhibition site.

• Cytochrome reductase is inhibited by the antibiotic antimycin A, which inhibits electron transfer at the Cyt b level.

• Antimycin A inhibition can overcome by adding ascorbate (vitamin C, a reducing agent), which directly reduces Cyt c, a step farther along the chain.

• Cytochrome oxidase is inhibited by cyanide (-CN-), azide (-N3), an carbon monoxide (CO); cyanide and azide complex with cytochrome heme groups in cytochromes a and a3 and prevent elctron transfer, carbon monoxide binds to cytochrome oxidase

Because oxidation of NADH and phosphorylation of ATP are separate biochemical reactions linked only by a PMF, substance that carry H+ across the mitochondrial inner membrane dissipate the pH gradient required for ATP synthesis

• These substances are called uncouplers because they uncouple or dissociate the oxidation of NADH (which still can continue) from the phosphorylation of ADP (which they inhibit).

a .Examples of uncouplers, substances that increase the H+ permeability of the inner mitochondrial membrane, are 2, 4-dinitrophenol and dicumarol.

• Uncoupling of NADH oxidation from ADP phosphorylation is a mechanism to generate heat in hibernating and newborn animals include humans.