There are three phases of respiration :
(1) External respiration : It is the exchange of respiratory gases (O2 and CO2) between an organism and its environment.
(2) Internal or Tissue respiration : Exchange of respiratory gases between tissue and extra cellular environment .
Both the exchange of gases occur on the principle of diffusion.
(3) Cellular respiration : It is an enzymatically-controlled stepped chemical process in which glucose is oxidised inside the mitochondria to produce energy-rich ATP molecules with high-energy bonds.
So, respiration is a biochemical process.

In respiration many types of high energy compounds are oxidised. These are called respiratory substrate or respiratory fuel and may include carbohydrates, fats and protein.

# (1) Carbohydrate : Carbohydrates such as glucose, fructose (hexoses), sucrose (disaccharide) or starch, insulin, hemicellulose (polysaccharide) etc; are the main substrates. Glucose are the first energy rich compounds to be oxidised during respiration. Brain cells of mammals utilized only glucose as respiratory substrate. Complex carbohydrates are hydrolysed into hexose sugars before being utilized as respiratory substrates. The energy present in one gram carbohydrate is – 4.4 Kcal or 18.4 kJ.

# (2) Fats : Under certain conditions (mainly when carbohydrate reserves have been exhausted) fats are also oxidised. Fat are used as respiratory substrate after their hydrolysis to fatty acids and glycerol by lipase and their subsequent conversion to hexose sugars. The energy present in one gram of fats is 9.8 Kcal or 41kJ, which is maximum as compared to another substrate.
The respiration using carbohydrate and fat as respiratory substrate, called floating respiration (Blackmann).

# (3) Protein : In the absence of carbohydrate and fats , protein also serves as respiratory substrate. The energy present in one gram of protein is : 4.8 Kcal or 20 kJ. when protein are used as respiratory substrate respiration is called protoplasmic respiration.

Organism can be grouped into following four classes on the basis of their respiratory habit -

# (1)Obligate aerobes : These organisms can respire only in the presence of oxygen. Thus oxygen is essential for their survival.
# (2) Facultative anaerobes : Such organisms usually respire aerobically (i.e., in the presence of oxygen) but under certain condition may also respire anaerobically (e.g., Yeast, parasites of the alimentary canal).
# (3) Obligate anaerobes : These organism normally respire anaerobically which is their major ATP- yielding process. Such organisms are in fact killed in the presence of substantial amounts of oxygen (e.g., Clostridium botulinum and C. tetani).
# (4) Facultative aerobes : These are primarily anaerobic organisms but under certain condition may also respire aerobically.

On the basis of the availability of oxygen and the complete or incomplete oxidation of respiratory substrate, the respiration may be either of the following two types :
1. Aerobic respiration
2.Anaerobic respiration

# Aerobic respiration : It uses oxygen and completely oxidises the organic food mainly carbohydrate (Sugars) to carbon dioxide and water. It therefore, releases the entire energy available in glucose.

- It is divided into two phases : Glycolysis, Aerobic oxidation of pyruvic acid

Glycolysis is a metabolic pathway that takes place in the cytosol of cells in all living organisms. This pathway can function with or without the presence of oxygen. In humans, aerobic conditions produce pyruvate and anaerobic conditions produce lactate. In aerobic conditions, the process converts one molecule of glucose into two molecules of pyruvate (pyruvic acid), generating energy in the form of two net molecules of ATP. Four molecules of ATP per glucose are actually produced, however, two are consumed as part of the preparatory phase. The initial phosphorylation of glucose is required to increase the reactivity (decrease its stability) in order for the molecule to be cleaved into two pyruvate molecules by the enzyme aldolase. During the pay-off phase of glycolysis, four phosphate groups are transferred to ADP by substrate-level phosphorylation to make four ATP, and two NADH are produced when the pyruvate are oxidized. The overall reaction can be expressed this way:

[Glucose + 2 NAD+ + 2 Pi + 2 ADP → 2 pyruvate + 2 NADH + 2 ATP + 2 H+ + 2 H2O + heat]

Starting with glucose, 1 ATP is used to donate a phosphate to glucose to produce glucose 6-phosphate. Glycogen can be converted into glucose 6-phosphate as well with the help of glycogen phosphorylase. During energy metabolism, glucose 6-phosphate becomes fructose 6-phosphate. An additional ATP is used to phosphorylate fructose 6-phosphate into fructose 1,6-disphosphate by the help of phosphofructokinase. Fructose 1,6-diphosphate then splits into two phosphorylated molecules with three carbon chains which later degrades into pyruvate.

# Glycolysis can be literally translated as "sugar splitting".

Aerobic respiration is completed in three steps -
(1) Oxidative decarboxylation/ Formation of acetyl CoA.
(2) Kreb's cycle/TCA cycle/Citric acid cycle.
(3) Electron transport system

Pyruvate is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase complex (PDC). The PDC contains multiple copies of three enzymes and is located in the mitochondria of eukaryotic cells and in the cytosol of prokaryotes. In the conversion of pyruvate to acetyl-CoA, one molecule of NADH and one molecule of CO2 is formed.

This is also called the Krebs cycle or the tricarboxylic acid cycle. When oxygen is present, acetyl-CoA is produced from the pyruvate molecules created from glycolysis. When oxygen is present, the mitochondria will undergo aerobic respiration which leads to the Krebs cycle. However, if oxygen is not present, fermentation of the pyruvate molecule will occur. In the presence of oxygen, when acetyl-CoA is produced, the molecule then enters the citric acid cycle (Krebs cycle) inside the mitochondrial matrix, and is oxidized to CO2 while at the same time reducing NAD to NADH. NADH can be used by the electron transport chain to create further ATP as part of oxidative phosphorylation. To fully oxidize the equivalent of one glucose molecule, two acetyl-CoA must be metabolized by the Krebs cycle. Two waste products, H2O and CO2, are created during this cycle.

The citric acid cycle is an 8-step process involving different enzymes and co-enzymes. During the cycle, acetyl-CoA (2 carbons) + oxaloacetate (4 carbons) yields citrate (6 carbons), which is rearranged to a more reactive form called isocitrate (6 carbons). Isocitrate is modified to become α-ketoglutarate (5 carbons), succinyl-CoA, succinate, fumarate, malate, and, finally, oxaloacetate.

The net gain of high-energy compounds from one cycle is 3 NADH, 1 FADH2, and 1 GTP; the GTP may subsequently be used to produce ATP. Thus, the total yield from 1 glucose molecule (2 pyruvate molecules) is 6 NADH, 2 FADH2, and 2 ATP.

In eukaryotes, oxidative phosphorylation occurs in the mitochondrial cristae. It comprises the electron transport chain that establishes a proton gradient (chemiosmotic potential) across the boundary of inner membrane by oxidizing the NADH produced from the Krebs cycle. ATP is synthesized by the ATP synthase enzyme when the chemiosmotic gradient is used to drive the phosphorylation of ADP. The electrons are finally transferred to exogenous oxygen and, with the addition of two protons, water is formed.

The electron transmitter system is also called electron transport chain (ETC), or cytochrome system (CS), as four out of these seven carriers are cytochrome. It is the major source of cells energy, in the respiratory breakdown of simple carbohydrates intermediates like phosphoglyceraldehyde, pyruvic acid, isocitric acid, ketoglutaric acid, succinic acid and malic acid are oxidised. The oxidation in all these brought about by the removal of a pair of hydrogen atoms (2H) from each of them. This final stage of respiration is carried out in ETS, located in the inner membrane of mitochondria (in prokaryotes the ETS is located in mesosomes of plasma membrane). The system consists of series of precisely arranged seven electron carriers (coenzyme) in the inner membrane of the mitochondrion, including the folds or cristae of this membrane. These seven electron-carriers function in a specific sequence and are : Nicotinamide adenine dinucleotide (NAD), Flavin mononucleotide (FMN), Flavin adenine dinucleotide (FAD), Co-enzyme-Q or ubiquinone, Cytochrome-b, Cytochrome-c, Cytochrome-a and Cytochrome-a3

# Five complex theory : According to Hatefi, (1976), Complex I to Complex IV are related to the electron transport.
 Complex V related to mainly with ATP synthesis, so it is called ATPase /ATP syntheses complex.
 The head piece (F1) of the oxysome consists of 5 hydrophobic subunits ( ), which are responsible for ATPase functioning.
 The stalk (F0) contain F5 (oligomycin sensitivity conferring protein) i.e. CSCP and F6. F0 are related to the proton channel and embeded fully in thickness of inner mitochondrial membrane.
 Five complex i.e. I, II, III, IV, V, have been isolated from mitochondrial membrane by chemical treatment.
 Complex I : NADH/NADPH : CoQ reductase
Complex II : Succinate : CoQ reductase
Complex III : Reduced CoQ (CoQH2) : cytochrome C reductase
Complex IV : Cytochrome C oxidase
Complex V : ATPase
# The first carrier in the chain is a flavoprotein which is reduced by NADH2. Coenzyme passes these electron to the cytochromes arranged in the sequence of b-c-a-a3, finally pass the electron to molecular oxygen. In this transport, the electrons tend to flow from electro-negative to electro-positive system, so there is a decrease in free energy and some energy is released so amount of energy with the electrons goes on decreasing. During electron-transfer, the electron-donor gets oxidised, while electron-acceptor gets reduced so these transfers involve redox-reaction and are catalysed by enzymes, called reductases. Oxidation and reduction are complimentary. This oxidation-reductiion reaction over the ETC is called biological oxidation.

During the electron transfers, the energy released at some steps is so high that ATP is formed by the phosphorylation of ADP in the presence of enzyme ATP synthetase present in the head of F1-particles present on the mitochondrial crista. This process of ATP synthesis during oxidation of coenzyme is called oxidative phosphorylation, so ETS is also called oxidative phosphorylation pathways.

From the cytochrome a3, two electrons are received by oxygen atom which also receives two proton (H+) from the mitochondrial matrix to form water molecule. So the final acceptor electrons is oxygen. So the reaction H2 + O2 ---> H2O (called metabolic water) is made to occur in many steps through ETC, so the most of the energy can be derived into a storage and usable form.

# Two route systems of ETC : The pairs of hydrogen atoms from respiratory intermediates are received either by NAD+ or FAD coenzymes which becomes reduced to NADH2 and FADH2. These reduced coenzyme pass the electrons on to ETC. Thus, regeneration of NAD+ or FAD takes place in ETC. There are two routes ETC :

- (a) Route 1 : NADH2 passes their electrons to Co-Q through FAD . In route 1 FAD is the first electron carrier. 3 ATP molecules are produced during the transfer of electron on following steps :
NAD to FAD
Cyt b to Cyt c and
Cyt a to Cyt a3
- (b) Route 2 : FADH2 passes their electron directly to FAD. 2 ATP molecules are produced during the transfer of electron on following steps.
Cyt b to Cyt c and
Cyt a to Cyt a3

# Development of proton gradient : At each step of ETC, the electron- acceptor has a higher electron –affinity than the electron-donor. The energy from electron-transport is used to move the proton (H+) from the mitochondrial matrix to inter-membranous or outer chamber. Three pairs of protons are pushed to outer chamber during the movement of electrons along route I while two pairs of protons are moved to outer chamber during the movement of electrons along route–II.
This generates a pH-gradient across the inner mitochondrial membrane with protons (H+) concentration higher in the outer chamber than in the mitochondrial matrix. This difference in H+ concentration across the inner mitochondrial membrane is called proton-gradient
( pH). Due to proton gradient, an electrical potential is developed across the inner mitochondrial membrane as the matrix is now electronegative with respect to the intermembranous (outer) chamber. The proton gradient and membrane electric potential collectively called proton motive force.

# (b) Proton flow : Due to proton-gradient, the protons returns to the matrix while passing through proton channel of F0-F1 ATPase. This proton gradient activates the enzyme ATP synthetase or F0 – F1 ATPase
ATP synthetase controls the formation of ATP from ADP and inorganic phosphate in the presence of energy.

Glycolysis occurs in the cytoplasm outside the mitochondrion in which 2NADH2 molecules are produced but ETC is located along inner mitochondrial membrane, so NADH2 of glycolysis must enter inside the mitochondrion to release energy. But the inner mitochondrial membrane is impermeable to NADH2. In mitochondrial membrane, there are 2 shuttle-system, each formed of carrier-molecule.

# These shuttle systems are :
(a) Malate-Aspartate shuttle
(b) Glycerol-Phosphate shuttle

It is more efficient and results in the transfer of electron from NAD. 2H in cytosol to NAD inside the mitochondrion, via NAD. 2H dehydrogenase as follows :
Electrons are transferred from NAD. 2H in cytosol to malate which traverses the inner mitochondrial membrane and reoxidised to form NAD. 2H thus resulting in the formation of oxaloacetate . Oxaloacetate does not readily cross the inner mitochondrial membrane and so a transamination reaction is needed to form aspartate which does traverse this barrier. As a result 3 ATP molecules are generated for each pair of electrons. Thus if this shuttle is predominant there is a gain of 38 ATP molecules by complete oxidation of one molecule of glucose.

It is less efficient and results in the reduction of FAD inside the mitochondrion.
If this shuttle predominates the electrons from NAD. 2H are transferred to FAD inside the mitochondrion as follows. NAD. 2H reacts with dihydroxyacetone phosphate (DHAP) in cytosol to form glycerol phosphate which diffuses through outer mitochondrial membrane to the outer surface of inner membrane. There glycerol phosphate reacts with membrane dehydrogenase to form dihydroxyacetone phosphate (DHAP) which returns to cytosol. In this process FAD is reduced to FADH2. Electrons from FADH2 directly pass to Q and other components of ETC and results in the synthesis of 2 ATP for each molecule of FADH2. In this case complete oxidation of glucose will result in a gain of 36 ATP molecule.

# Which shuttle predominates depends on the particular species and tissues envolved, for example : 38 ATP are formed in kidney, heart and liver cell while 36 ATP molecules are formed in muscle cells and nerve cells. In these cells glycerol-phosphate shuttle is predominant and 2 ATP formed from NADH2.

Without oxygen, pyruvate (pyruvic acid) is not metabolized by cellular respiration but undergoes a process of fermentation. The pyruvate is not transported into the mitochondrion, but remains in the cytoplasm, where it is converted to waste products that may be removed from the cell. This serves the purpose of oxidizing the electron carriers so that they can perform glycolysis again and removing the excess pyruvate. Fermentation oxidizes NADH to NAD+ so it can be re-used in glycolysis. In the absence of oxygen, fermentation prevents the buildup of NADH in the cytoplasm and provides NAD+ for glycolysis. This waste product varies depending on the organism. In skeletal muscles, the waste product is lactic acid. This type of fermentation is called lactic acid fermentation. In strenuous exercise, when energy demands exceed energy supply, the respiratory chain cannot process all of the hydrogen atoms joined by NADH. During anaerobic glycolysis, NAD+ regenerates when pairs of hydrogen combine with pyruvate to form lactate. Lactate formation is catalyzed by lactate dehydrogenase in a reversible reaction. Lactate can also be used as an indirect precursor for liver glycogen. During recovery, when oxygen becomes available, NAD+ attaches to hydrogen from lactate to form ATP. In yeast, the waste products are ethanol and carbon dioxide. This type of fermentation is known as alcoholic or ethanol fermentation. The ATP generated in this process is made by substrate-level phosphorylation, which does not require oxygen.

Fermentation is less efficient at using the energy from glucose: only 2 ATP are produced per glucose, compared to the 38 ATP per glucose nominally produced by aerobic respiration. This is because the waste products of fermentation still contain chemical potential energy that can be released by oxidation. Ethanol, for example, can be burned in an internal combustion engine like gasoline. Glycolytic ATP, however, is created more quickly. For prokaryotes to continue a rapid growth rate when they are shifted from an aerobic environment to an anaerobic environment, they must increase the rate of the glycolytic reactions. For multicellular organisms, during short bursts of strenuous activity, muscle cells use fermentation to supplement the ATP production from the slower aerobic respiration, so fermentation may be used by a cell even before the oxygen levels are depleted, as is the case in sports that do not require athletes to pace themselves, such as sprinting.