Research

Our field of interest is the respiratory complex I from mitochondria and bacteria. This enzyme (systematic name: NADH:Ubiquinone Oxidoreductase, E.C. 1.6.5.3) is the first complex of cell respiration (respiratory chain). It takes up electrons from the water soluble electron carrier NADH and transfers them to ubiquinone, a hydrophobic compound being able to diffuse within the inner mitochondrial membrane. From ubiquinone, electrons are transferred to the other complexes of the respiratory chain. The energy released by this process is used by the enzyme to pump hydrogen ions from the inner to the outer side of the membrane. In this way, the energy is available as an electrochemical membrane potential, which can be used up by other enzymes, e.g. the ATP synthase or transport proteins.

The respiratory complex I is found in the membranes of mitochondria from most eukaryotes as well as in many bacteria, but hasn´t been found in archaebacteria until now. The complex is made up of three different modules, related to some transporter proteins (e.g. a K+/H+ antiporter), hydrogenases (NiFe hydrogenase) and diaphorases. The minimum number of subunits needed for the functional enzyme was shown to be 14 in bacteria, in higher organisms (e.g. bos taurus), however, complex I is made up of up to 46 subunits. The total mass of the enzyme ranges from 500 to 1000 kDa, depending on the organism. For this reason, the complex belongs to the biggest enzymes at all. One flavin mononucleotide and a set of iron-sulfur clusters have been identified to be cofactors of the enzyme. Other cofactors are assmumed but haven´t been detected yet. By means of electron microscopy a rough picture of the enzyme could be obtained at a resolution of about 30 A. The complex resembles the letter “L”. One arm of the “L” extends into the inner part of the mitochondria and bacteria, respectively, the other arm lies within the membrane. Further information on the enzyme can be obtained at the complex I homepage.

Further Informations: Phylogeny of Complex I

Archaebacteria

Archaebacteria make up the third branch in the sytem of living organisms (besides bacteria and eukaryotes). Like bacteria, they don´t have cell organells. However, some metabolism paths as well as the organisation of the genome are similar to eukaryotes. Many archaebacteria live under extreme conditions, such as high temperatures (80 to 100°C) or extreme pressure (e.g. 20 MPa at seabed). Archaebacteria are counted among prokaryotes.

Respiratory chain

The respiratory chain is the last step in the degrading metabolism (katabolism) of all aerobic organisms. Reduction equivalents (mainly NADH) obtained from glycolysis and the citrate cycle are used for the reduction of oxygen, producing water. The energy from this reaction is stored as an electrochemical membrane potential. It is used up by a set of cell processes, e.g. flagellar movement in bacteria, transport of substances across a membrane as well as the synthesis of ATP.

ATP synthase

ATP, the universal “energy currency” of the cell, is synthesized by ATP synthase. It joins inorganic phosphate to ADP under separation of water, so that an anhydride is formed. The energy needed for this reaction to occur is provided by the backflow of protons previously having been pumped by the respiratory complexes to the outer side of the membrane. The flow of electrons thereby drives a rotation of the enzyme, not unlike an electric motor. The rotation was directly observed on a molecular level by a japanese group (Noji, H., Yasuda, R., Yoshida, M. & Kinosita, K. Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature 386, 299–302.).

Bacteria

Bacteria are single-celled microorganisms, differing from eukaryotes in lacking cell organells. As they don´t have a cell nucles, they are counted among prokaryotes. They contain a singular chromosome and thy cytoplasm, surrounded by a membrane. Furthermore, they contain a cell wall.

Diaphorase

Diaphorases are a set of enzymes oxidizing NADH or reducing NAD+.

E.C.

E.C. stands for “Enzyme Classification”, a system to name enzymes after their function. The numbers are described here.

Iron sulfur clusters

Iron sulfur clusters are cofactors built of iron and sulfur atoms, bound to a protein via the amino acids cysteine or histidine. According to the number of the iron and sulfur atoms, they are termed [2Fe2S], [3Fe4S] and [4Fe4S] clusters, respectively. These cofactors are taking part in electron transport, catalysis, structure stabilization and regulation of metabolism.

Example of an iron sulfur cluster:

Electrochemical membrane potential

As can be guessed by the name, the electrochemical membrane potential is made up of two components: an electric one, caused by an uneven distribution of charge carriers (both positive and negative) on both sides of the mebrane, and a chemical one, caused by an uneven distribution of chemical compounds or ions. Both have the tendency to distribute evenly, but are hindered to do so by the membrane. The energy stored in the electrochemical membrane potential is used up by the transport of substances across a membrane, for ATP synthesis or flagella rotation. The electrochemical membrane potential is described by the formula:

Eukaryotes:

Eukaryotes contain both a cell nucleus and cell organells, e.g. vesicula, the endoplasmatic reticulum and the Golgi apparatus. Furthermore, they contain mitochondria, the “power plants of the cell”, providing the main part of the energy of the metabolism. Among the eukaryotes are counted protozoae, fungi, higher plants and animals.

Flavine mononucleotide

The flavine mononucleotide is a cofactor consisting of a flavine system, a ribose part and a phosphate part. It can exist oxidized (FMN), as well as reduced (FMNH2).

Hydrogenases

Hydrogenases are enzymes being able to reduce protons to molecular hydrogen. They are found in anaerobic organisms, e.g. bacteria, living in the intestinal tract of ruminants.

Mitochondria

Mitochondria are the “power plants” of the cell. They only exist in eukaryotes. The citrate cycle, the reactions of the respiratory chain as well as a set of other metabolic recations occur in mitochondria. They contain two shell membranes and their own circular genome, so it is believed that mitochondria are former bacteria having been taken up from other cells in the course of evolution. The inner part of mitochondria is called matrix, the space between both membranes as intramembranous space. The mitochondrial genome is propagated independently, the information contained within it used for the synthesis of proteins.

Prokaryotes

All species not containing a cell nucleus are counted among prokaryotes. Of the three domains in the tree of life (bacteria, archaebacteria and eukaryotes) bacteria and archaebacteria belong to prokaryotes.

Transport proteins

Transport proteins catalyze the transport of chemical compounds (also proteins) across a membrane. So, strictly speaking, they don´t count among enzymes, since the transported compound isn´t changed in a chemical way. In other respects, however, the transport reaction can be regarded as an enzymatic reaction. In particular it follows Michaelis-Menten kinetics. There are transport proteins that facilitate diffusion (transport facilitators), transporters that carry substances at the same time in the same direction (symporters) or in opposite direction (antiporters) and transporters that use up ATP in the transport reaction. This last case is known as secondary transport.