The energy generating machinery of cells:

1. The energy generating machinery of cells: Mitochondria

2. Basic structure • Tubular or filamentous organelles – the largest organelle in most cells following the nucleus. (about 1 by 5-10 microns). They accumulate in the more active areas of cells ex. Base of renal tubular cell or apex of intestinal cell. • Surrounded by a double membrane (i.e. 2 membranes, each a phospholipid bi-layer). The inner membrane has many invaginations- cristae • This results in 2 spaces, an inner (or intercristal) mitochondrial space and an outer or intra-cristal space. The intercristal space contains the Matrix.

4. Structure II • Inside the matrix or on the are a few ribosomes, RNA, mitochondrial DNA, and electron-dense granules rich in cations. Also present are the large enzymes complexes required for fatty acid oxidation and the Kreb’s cycle. • On the inner mitochondrial membrane, are located the proteins of oxidative phosphorylation and the electron transfer chain. About 75% of the membrane is protein – highest %content for any eukaryotic cell membrane. • In a typical liver mitochondrion, the inner membrane has a surface area 5 times the outer.

5. Structure III • Mitochondria grow and divide to produce new mitochondria. They are also digested to re-enter the metabolite pool inside the cell. • Some recent studies looking at numerous electron micrographs of the same cell suggest that the mitochondria may actually be part of a budding, continuous network all over the cell ( like the ER) rather than discreet organelles.

6. Cellular differences • The amount of mitochondria per cell differs. Hepatocytes can have about 2000. The amount is characteristic for each cell type. • Mitochondria differ in quality as well as quantity in different cells. In muscle, heart and kidney, organs with a higher metabolic rate, there are, correspondingly, more numerous cristae. Ex. In the heart they have 3 times as many as in liver. In steroid-producing cells, they have tubular cristae instead of the usual flat shelf-like cristae. • In brown fat, mitochondria are also functionally different.

8. Functional division of compartments. • Outer membrane –phospholipid synthesis, fatty acid desaturation, fatty acid elongation. • Intermembrane space- nucleotide phosphorylation • Inner membrane (the essential function) –Electron transport, oxidative phosphorylation, metabolite transport in/out. • Matrix – Citric acid cycle, b-oxidation of fatty acids, DNA replication, RNA synthesis, protein sysnthesis.

9. Function. • Basic function – to generate energy. With mitochondria, the cell generates 15 times more energy than with glycolysis alone. The mechanism is dependant on selectively permeable membranes (cristae). This is largely what allowed Eukaryotes to develop as extensively as they have done – bacteria can only use their cell membranes and are not as capable at oxidative phosphorylation. • Basic principle is the use of energy in the molecules of food (via the carriers of the electron transport chain which carry this energy in the form of high energy electrons) to create a H+ gradient across the inner mitochondrial membrane. This gradient is then used to convert ADP into ATP.

10. ATP Synthase ATP ADP+Pi Mechanism of energy production porins Inter-membrane space Rotor H+ H+ H+ Stator 3H+ e` e` Q Cyt c 3H+ +O2 H+ H+ H+ Cytochrome oxidase complex NADH Dehydrogenase complex NAD+ NADH Cytochrome b-c1 complex H2O Matrix

11. Function II • The potential difference in the transfer of a high energy electron from NADH to Oxygen is about 1.14Volts/Mole. If this were released in one single reaction, the energy would be almost explosive and mostly lost to the environment as heat. • By slowly reducing the energy of the electron along the electron transport chain and using it to pump protons, almost half the energy is used to convert ADP to ATP. About 3-4 protons moving back across the ATPsynthase are needed to form one molecule of ATP. • Therefore, chemical energy is transferred into an electrochemical gradient (pH and H+ concentration gradient), this is then transferred into kinetic (rotational) energy, and this is again transferred into chemical energy in ATP.

12. Structural adaptations to function. • Much depends on the impermeability of the inner mitochondrial membrane to even very small ions such that transfer is dependant on proteins spanning the membrane, in a regulated fashion. – This inner membrane contains 20% of a mitochondrial specific phospholipid (cardiolipin) which is a kind of double phospholipid with 4 (as opposed to 2) fatty acid chains. This makes it more impermeable. • In brown fat, mitochondria have a natural uncoupling agent – which allows transfer of H from NADH to O2 (along the chain) without generation of an electro-chemical proton gradient or ATP and with the resultant production of heat. This is a membrane protein which acts as a proton channel, thus destroying the H+ gradient.

13. Mitochondrial genome • Mitochondria thought to be evolutionary prokaryotes endocytosed and which developed a symbiotic relationship with Eukaryotes. Some few (evolutionarily distant) eukaryotes do not have mitochondria. ( Eg Giardia – protozan) • Mitochondrial genome – circular DNA – like bacteria. Codes for only about 11 proteins. Also rRNA and all tRNAs. Most of other mitochondrial genes have been transferred to the cell nucleus. Mitochondrial DNA replicates separately from the cell cycle. • Genome divided up amongst daughter mitochondria when they divide. (may have 1-15 copies)

14. Peculiarities. • Mitochondria only import proteins. They do not export any. – Makes sense – small genome. Important – co-ordination with nuclear genes in mitochondrial reproduction/growth. • Mitochondrial Ribosomes – resistant to antibiotics which effect all eukaryotic ribosomes – eg cyclohexamide – because it is Prokaryotic type • Genetic code is different. Only one tRNA per AA. (Non-ambiguous). Wobble to allow different codon reading. UGA ( usually stop) reads Tryptophan, AGA, AAG (usually arginine) read STOP, AUA (usually isoleucine) reads methionine.

15. Non-mendelian inheritance • Mitochondria divided amongst daughter cells. Therefore mitochondrial genes are not inherited in the usual way of meiosis. Chance can distribute all abnormal genomes to one daughter cell and none to another. • Also, in mammals, due to cytoplasm coming mostly from mother, any mitochondrial anomaly will always be transferred by the mother. Will all her grandchildren have the disease or not?? What about the sons of her daughters? What about the daughters of her sons?

16. Clinical Correlates • Cyanide –(Zyclon B in WWII)- blocks oxidative phosphorylation in mitochondria. Antidotes (which work imperfectly) include dicobalt EDTA or hydroxycobalamin which bind the cyanide strongly. • Analysis of mutations in the mitochondrial genome have helped determine the origin of the different races and populations. The largest range of mutations occurs in African populations. All the other races are more similar than the different African ones, are between themselves indicating that we all evolved out of Africans, the earliest humans • Mitochondrial anomalies in all mitochondria would usually be lethal. However due to the possibility of mutations having developed in only some part of the mitochondrial population of the oocyte (some 300,000 mitochondria) different amounts of defective organelles will reach different tissues in the resultant embryo. The severity of the disease will also depend on how the normal and abnormal mitochondria have been distributed. Because of this, mitochondrial diseases like ‘Myoclonic epilepsy and ragged red fibre disease’ have a marked variability between individuals due to different extents of involvement of diverse tissues.

17. Clinical correlates II • Such mitochondrial diseases will effect tissues most dependant on high ATP levels such as muscle and nervous tissue. However, as can be surmised, a child may appear normal since most mitochondria in the muscle and nerve happen to be normal, and her daughter may be very ill. Or vice versa. • Mitochondria are the seat of apoptosis – targetted cell death - through the release of cytochrome c • The clinical picture of the diverse mitochodrial disease, includes this important triad: - Audiologic anomalies Neuromuscular setbacks following infections Multi-organ involvement