Большая часть дыхания осуществляется в митохондриях. Mitochondria have a double membrane: the outer membrane contains many protein channels called porins, which let almost any small molecule through; while the inner membrane is more normal and is impermeable to most materials. The inner membrane is highly folded into folds called christae, giving a larger surface area. The electron microscope reveals blobs on the inner membrane, which were originally called stalked particles. These have now been identified as the enzyme complex that synthesises ATP, are is more correctly called ATP synthase (more later). the space inside the inner membrane is called the matrix, and is where the Krebs cycle takes place. The matrix also contains DNA, tRNA and ribosomes, and some genes are replicated and expressed here.

Details of Respiration

1. Glucose enters cells from the tissue fluid by passive transport using a specific glucose carrier. This carrier can be controlled (gated) by hormones such as insulin, so that uptake of glucose can be regulated.

2. The first step is the phosphorylation of glucose to form glucose phosphate, using phosphate from ATP. Glucose phosphate no longer fits the membrane carrier, so it can’t leave the cell. This ensures that pure glucose is kept at a very low concentration inside the cell, so it will always diffuse down its concentration gradient from the tissue fluid into the cell. Glucose phosphate is also the starting material for the synthesis of pentose sugars (and therefore nucleotides) and for glycogen.

3. Glucose is phosphorylated again (using another ATP) and split into two triose phosphate (3 carbon) sugars. From now on everything happens twice per original glucose molecule.

4. The triose sugar is changed over several steps to form pyruvate, a 3-carbon compound. In these steps some energy is released to form ATP (the only ATP formed in glycolysis), and a hydrogen atom is also released. This hydrogen atom is very important as it stores energy, which is later used by the respiratory chain to make more ATP. The hydrogen atom is taken up and carried to the respiratory chain by the coenzyme NAD, which becomes reduced in the process.

(oxidised form O) NAD + H O NADH (< reduced form)

Pyruvate marks the end of glycolysis, the first stage of respiration. In the presence of oxygen pyruvate enters the mitochondrial matrix to proceed with aerobic respiration, but in the absence of oxygen it is converted into lactate (in animals and bacteria) or ethanol (in plants and fungi). These are both examples of anaerobic respiration. Pyruvate can also be turned back into glucose by reversing glycolysis, and this is called gluconeogenesis.

5. Once pyruvate has entered the inside of the mitochondria (the matrix), it is converted to a compound called acetyl coA. Since this step is between glycolysis and the Krebs Cycle, it is referred to as the link reaction. In this reaction pyruvate loses a CO₂ and a hydrogen to form a 2-carbon acetyl compound, which is temporarily attached to another coenzyme called coenzyme A (or just coA), so the product is called acetyl coA. The CO₂ diffuses through the mitochondrial and cell membranes by lipid diffusion, out into the tissue fluid and into the blood, where it is carried to the lungs for removal. The hydrogen is taken up by NAD again.

6. The acetyl CoA then enters the Krebs Cycle, named after Sir Hans Krebs, who discovered it in the 1940s at Leeds University. It is one of several cyclic metabolic pathways, and is also known as the citric acid cycle or the tricarboxylic acid cycle. The 2-carbon acetyl is transferred from acetyl coA to the 4-carbon oxaloacetate to form the 6-carbon citrate. Citrate is then gradually broken down in several steps to re-form oxaloacetate, producing carbon dioxide and hydrogen in the process. As before, the CO₂ diffuses out the cell and the hydrogen is taken up by NAD, or by an alternative hydrogen carrier called FAD. These hydrogens are carried to the inner mitochondrial membrane for the final part of respiration.

The Respiratory Chain

The respiratory chain (or electron transport chain) is an unusual metabolic pathway in that it takes place within the inner mitochondrial membrane, using integral membrane proteins. These proteins form four huge trans-membrane complexes called complexes I, II, II and IV. The complexes each contain up to 40 individual polypeptide chains, which perform many different functions including enzymes and trans-membrane pumps. In the respiratory chain the hydrogen atoms from NADH gradually release all their energy to form ATP, and are finally combined with oxygen to form water.

1. NADH molecules bind to Complex I and release their hydrogen atoms as protons (H⁺) and electrons (e^-). The NAD molecules then returns to the Krebs Cycle to collect more hydrogen. FADH binds to complex II rather than complex I to release its hydrogen.

2. The electrons are passed down the chain of proteins complexes from I to IV, each complex binding electrons more tightly than the previous one. In complexes I, II and IV the electrons give up some of their energy, which is then used to pump protons across the inner mitochondrial membrane by active transport through the complexes. Altogether 10 protons are pumped across the membrane for every hydrogen from NADH (or 6 protons for FADH).

3. In complex IV the electrons are combined with protons and molecular oxygen to form water, the final end-product of respiration. The oxygen diffused in from the tissue fluid, crossing the cell and mitochondrial membranes by lipid diffusion. Oxygen is only involved at the very last stage of respiration as the final electron acceptor, but without the whole respiratory chain stops.

4. The energy of the electrons is now stored in the form of a proton gradient across the inner mitochondrial membrane. It’s a bit like using energy to pump water uphill into a high reservoir, where it is stored as potential energy. And just as the potential energy in the water can be used to generate electricity in a hydroelectric power station, so the energy in the proton gradient can be used to generate ATP in the ATP synthase enzyme. The ATP synthase enzyme has a proton channel through it, and as the protons "fall down" this channel their energy is used to make ATP, spinning the globular head as they go. It takes 4 protons to synthesise 1 ATP molecule.

This method of storing energy by creating a protons gradient across a membrane is called chemiosmosis, and was discovered by Peter Mitchell in the 1960s, for which work he got a Nobel prize in 1978. Some poisons act by making proton channels in mitochondrial membranes, so giving an alternative route for protons and stopping the synthesis of ATP. This also happens naturally in the brown fat tissue of new-born babies and hibernating mammals: respiration takes place, but no ATP is made, with the energy being turned into heat instead.

				John Walker was born in Halifax, Yorkshire, in 1941. Educated at Rastrick Grammar School and St Catherine’s College, Oxford, he received the BA degree in Chemistry in 1964. Then he began research on peptide antibiotics with E. P. Abraham in the Sir William Dunn School of Pathology, Oxford, and was awarded the DPhil degree in 1969. This was followed by a period of five years working abroad: from 1969-71 at the School of Pharmacy at the University of Wisconsin, and from 1971-74 in Paris. In 1974 he met Fred Sanger and Ieuan Harris at an EMBO workshop in Cambridge and shortly after moved to the Protein and Nucleic Acid Chemistry Division of the Laboratory of Molecular Biology in Cambridge. At this time, in the mid-1970s, Sanger was inventing his methods for sequencing DNA. They were applied first to the related bacteriophages fX174 and G4, and then to DNA from human and bovine mitochondria. Walker analysed the sequences of the proteins from G4 and from mitochondria using direct methods. This work led to the discovery of triple over-lapping genes in G4 (with D. Shaw and B. G. Barrell), and to the discovery that subunits I and II of cytochrome c oxidase were encoded in the DNA in mitochondria. He also helped to uncover the modified genetic code in mitochondria.
			(jointly with Paul Boyer and Jens Skou) "for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP).”
			Simplified Picture of ATP Synthase ATP synthase (F1Fo-ATPase) is the central enzyme in energy conversion in mitochondria, chloroplasts and bacteria. It uses a proton motive force, generated across the membrane by electron flow, to drive the synthesis of ATP from ADP and inorganic phosphate. The Fo part through which hydrogen ions (H+) stream is located in the membrane. The F1 part which synthesises ATP is outside the membrane. When the hydrogen ions flow through the membrane via the disc of c subunits in the Fo part, the disc imparts a twist to the g - subunit which protrudes from the F1 part and is attached to the disc. The three alpha and three beta subunits in the F1 part cannot rotate, however. They are locked in a fixed position by the b subunit, which in turn is anchored in the membrane. Thus the gamma subunit rotates inside the cylinder formed by the six alpha and beta subunits. Since the gamma subunit is asymmetrical it compels the beta subunits to undergo structural changes. This leads to the beta subunits binding ATP and ADP with differing strengths. The interconversion of these states, and hence the continuous production of ATP, occurs as the g subunit rotates.
				During the work on mitochondria, he developed an interest in the enzyme complexes in the inner membrane of the organelle that carry out oxidative phosphorylation, and in 1978 he began a structural study of the ATP synthase from bovine heart mitochondria. This work eventually resulted in a complete sequence analysis of the complex, and in an atomic resolution structure of the F1 catalytic domain of the enzyme. Walker was elected a Fellow of the Royal Society in 1995. He was awarded the Johnson Foundation Prize by the University of Pennsylvania in 1994, and the CIBA Medal and Prize of the Biochemistry Society in 1996. In 1997 he was elected a Fellow of Sidney Sussex College, Cambridge, and became an Honorary Fellow of St Catherine’s College, Oxford. He was knighted in 1999 and appointed to be Director of the Dunn Human Nutrition Unit.

How Much ATP is Made in Respiration?

We can now summarise respiration and see how much ATP is made from each glucose molecule. ATP is made in two different ways:

Some ATP molecules are made directly by the enzymes in glycolysis or the Krebs cycle. This is called substrate level phosphorylation (since ADP is being phosphorylated to form ATP).
Most of the ATP molecules are made by the ATP synthase enzyme in the respiratory chain. Since this requires oxygen it is called oxidative phosphorylation. Scientists don’t yet know exactly how many protons are pumped in the respiratory chain, but the current estimates are: 10 protons are pumped by NADH; 6 by FADH; and 4 protons are needed by ATP synthase to make one ATP molecule. This means that each NADH can make 2.5 ATPs (10/4) and each FADH can make 1.5 ATPs (6/4). Previous estimates were 3 ATPs for NADH and 2 ATPs for FADH, and these numbers still appear in most textbooks, although they are now know to be wrong.
Two ATP molecules are used at the start of glycolysis to phosphorylate the glucose, and these must be subtracted from the total.

The table below is an "ATP account" for aerobic respiration, and shows that 32 molecules of ATP are made for each molecule of glucose used in aerobic respiration. This is the maximum possible yield; often less ATP is made, depending on the circumstances. Note that anaerobic respiration (glycolysis) only produces 2 molecules of ATP.

Stage	molecules produced per glucose	Final ATP yield (old method)	Final ATP yield (new method)
Glycolysis	2 ATP used	-2	-2
	4 ATP produced (2 per triose phosphate)	4	4
	2 NADH produced (1 per triose phosphate)	6	5
Link Reaction	2 NADH produced (1 per pyruvate)	6	5
Krebs Cycle	2 ATP produced (1 per acetyl coA)	2	2
	6 NADH produced (3 per acetyl coA)	18	15
	2 FADH produced (1 per acetyl coA)	4	3
Total		38	32

Other substances can also be used to make ATP. Triglycerides are broken down to fatty acids and glycerol, both of which enter the Krebs Cycle. A typical triglyceride might make 50 acetyl CoA molecules, yielding 500 ATP molecules. Fats are a very good energy store, yielding 2.5 times as much ATP per g dry mass as carbohydrates. Proteins are not normally used to make ATP, but in times of starvation they can be broken down and used in respiration. They are first broken down to amino acids, which are converted into pyruvate and Krebs Cycle metabolites and then used to make ATP.