Mitochondria

Their name is derived from Greek mitos, "thread", and chondros, "grain" or "seed". In some cells mitochondria are long, almost filamentous, but in most others they are elliptical or spherical. In each liver cell of the rat there are perhaps 1000 mitochondria. They have a diameter of about 1 micrometer, close to the size of bacterial cells. Some types of eukaryotic cells contain only a few very large mitochondria (e.g., sperm cells or yeast cells), whereas others contain many thousands (e.g., egg cells). Each mitochondrion has two membrane systems. The outer membrane is smooth, surrounding the mitochondrion completely. The inner membrane has infoldings called cristae. The inner compartment of mitochondria is filled with gel-like matrix.

Mitochondria generally contain several copies of small circular DNA as well as RNA and ribosomes. Mitochondrial DNA codes for subunits of enzyme complexes involved in oxidative phosphorylation. One may well ask why mitochondria contain DNA. This question has led to the interesting concept that mitochondria originally arose during biological evolution by the invasion of cytoplasm of large anaerobic prokaryotic cells by smaller prokaryotes capable of using molecular oxygen to oxidize their nutrients within the host cells.

The mitochondria are the power plants of the cells. They contain many enzymes that together catalyze the oxidation of organic cell nutrients by molecular oxygen to yield carbon dioxide and water. Some of these enzymes are located in the matrix and some in the inner membrane. Much chemical energy is released during these oxidations, which is used to generate ATP, the major energy-carrying molecule of cells.

Oxidative Phosphorylation system

In mammalian mitochondria, this system is composed of five enzyme complexes. Those are NADH-quinone oxidoreductase (complex I), succinate-quinone oxidoreductase (complex II), Quinol-cytochrome c oxidoreductase (complex III or bc1 complex), Cytochrome c oxidase (complex IV), and ATP synthase (complex V).

Mitochondrial H+-translocating NADH-quinone oxidoreductase (complex I)

This enzyme complex is one of three energy-transducing complexes that constitute the respiratory chain in mammalian mitochondria. This NADH-Q oxidoreductase is the point of entry for majority of electrons that traverse the respiratory chain eventually resulting in the reduction of oxygen to water. In 1961, the mitochondrial H+-translocating NADH-Q oxidoreductase (complex I) was first isolated from bovine heart by Hatefi and coworkers. This enzyme complex is composed of at least 46 dissimilar subunits. As far as our present knowledge is concerned, complex I has the most intricate structure of the membrane-bound enzyme complex.

Why research on complex I is important

Research on complex I has recently taken on greater significance since the finding that many human mitochondrial diseases involve structural and functional defects at the level of this enzyme complex. Examples include a variety of neuromuscular diseases, including myoclonic epilepsy and ragged red fibers (MERRF); mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS); Chronic External Ophthalmoplegia Plus (CEOP); Kearns-Sayre syndrome (KSS). Many cases of Leber's hereditary optic neuropathy (LHON), appear to be associated with a defect in complex I. The defect identified in many cases is a single nucleotide change in the mitochondrial DNA converting the 340th amino acid of the ND4 subunit of complex I from an arginine to a histidine. In addition to these documented cases, the compound, 1-methyl-4-phenylpyridium, an inhibitor of complex I, produces idiopathic Parkinsonism in rats and human, which suggests a link between Parkinson's disease and function of mitochondrial complex I.

Use and choice of a bacterial model system

In spite of its structural complexity, the results of a strategy designed to investigate complex I directly have been reported. It is clear that this approach has met with limited success. Another approach is to find a simpler system in which the elucidation of the structural and functional relationships within complex I will also be simpler. Because bacterial respiratory enzyme complexes are known to be generally simpler in structure than their mitochondrial counterparts while retaining similarity to their mitochondrial counterparts in terms of electron carriers, we screened various bacterial species in terms of their NADH-Q oxidoreductases. In the course of this research, the various bacterial NADH-q oxidoreductases were found to fall into one of the following 3 categories: NDH-2, Na+-NDH, or NDH-1. Those enzyme complexes lacking an energy-coupling site are known as NDH-2 (NADH dehydrogenase 2). Those enzymes which bear an energy-coupling site are designated Na+-NDH, if they pump sodium ions, or NDH-1 (NADH dehydrogenase 1) if they function as H+ pumps.

Why Paracoccus denitrificans ?

Paracoccus denitrificans is a Gram-negative soil bacterium and has been called "a free-living mitochondrion" because of similarity in electron transfer components, similarity of 16S rRNA sequence between this bacterium and mitochondria, and similarities of amino acid sequences of various enzymes. Aerobically grown Paracoccus expresses a mammalian mitochondrial type respiratory chain which contains NDH-1. Therefore, the Paracoccus NDH-1 is a useful model for study of mitochondrial complex I.

Why Thermus thermophilus?

Thermus thermophilus, which was isolated from a hot spring in Japan, is an extremely thermophilic, obligatory aerobic, Gram-negative, and chemoheterotrophilic bacterium. The bacterium is capable of growing in the temperature range from 45C to 85C with optimum growth temperature of 70C. Its respiratory chain contains NDH-1. The thermostability of the NDH-1 is expected to provide a great advantage for structural studies of the NDH-1.