A conventional view of mitochondrial structure is represented at right. The respiratory chain is embedded in cristae of the inner membrane.
Spontaneous electron transfer through respiratory chain complexes I, III, & IV is coupled to H+ ejection from the matrix to the intermembrane space. Because the outer membrane contains large channels, protons in the intermembrane space equilibrate with the cytosol.
Respiration-linked pumping of protons out of the mitochondrial matrix conserves some of the free energy of spontaneous electron transfers as potential energy of an electrochemical H+ gradient.
3-D reconstructions based on electron micrographs taken with large depth of field at different tilt angles have indicated that the infoldings of the inner mitochondrial membrane are actually variable in shape and are connected to the periphery and to each other by narrow tubular regions.
At right is an electron micrograph, provided by Dr. Carmen Mannella of the Wadsworth Center, of a Neurospora mitochondrion in a frozen sample in the absence of fixatives or stains that might alter the appearance of internal structures.
Tubular cristae connect to the inner membrane via narrow passageways that may limit the rate of H+ equilibration between the lumen of cristae and the intermembrane space. See also Fig 22-4 in Voet & Voet, Biochemistry, 3rd Edition, p. 799, and a Wadsworth Center website.
A total of 10 protons are ejected from the mitochondrial matrix per 2 electrons transferred from NADH to oxygen via the respiratory chain. The H+/e- ratio for each respiratory chain complex will be discussed separately. (See also article by P. Hinkle).
Complex I (NADH Dehydrogenase) transports 4H+ out of the mitochondrial matrix per 2e- transferred from NADH to coenzyme Q.
Lack of high-resolution structural information for the membrane domain of complex I has hindered elucidation of the mechanism of H+ transport through this complex. Direct coupling of transmembrane proton flux and electron transfer is unlikely, because the electron-transferring prosthetic groups, FMN and iron-sulfur centers, are all located in the peripheral domain of complex I (see notes on electron transfer chain). Thus it is assumed that protein conformational changes are involved in H+ transport, as with an ion pump.
Complex III (bc1 complex): H+ transport in complex III involves coenzyme Q (CoQ). The "Q cycle" depends on the mobility of CoQ within the lipid bilayer. There is evidence for one-electron transfers, with an intermediate semiquinone radical state. Q Cycle:
Electrons enter complex III via coenzyme QH2, which binds at a site on the positive side of the inner mitochondrial membrane, adjacent to the intermembrane space. QH2 gives up one electron to the Rieske iron-sulfur center (Fe-S). Fe-S is reoxidized by transfer of the electron to cytochrome c1, which passes it out of the complex to cytochrome c. The loss of one electron from QH2 would generate a semiquinone radical, shown here as Q·-, although the semiquinone might initially retain a proton as QH·. A second electron is transferred from the semiquinone to cytochrome bL (heme bL), which passes it across the membrane via cytochrome bH (heme bH) to another CoQ bound at a site on the matrix side of the membrane. The fully oxidized CoQ, generated as the second electron is passed to the b cytochromes, may then dissociate from its binding site adjacent to the intermembrane space. Accompanying the two-electron oxidation of bound QH2, 2H+ are released to the intermembrane space.
Q cycle (one version)
In an alternative mechanism that has been proposed, the two electron transfers from QH2 to Fe-S & cyt bL may be essentially simultaneous, eliminating the semiquinone intermediate. (See the list of recent reviews for more detailed discussions of proposed mechanisms.)
It takes 2 cycles for CoQ, bound at the site near the matrix side of the membrane, to be reduced to QH2, as it accepts 2 electrons from the b hemes and 2 H+ are extracted from the matrix compartment. In 2 cycles, 2 QH2 enter the pathway, and one is regenerated.
Overall reaction catalyzed by complex III, including net inputs and outputs of the Q cycle: QH2 + 2H+(matrix side) + 2 cyt c (Fe3+) a Q + 4H+(outside) + 2 cyt c (Fe2+)
Per 2e- transferred through the complex to cytochrome c, 4H+ are released to the intermembrane space. While 4H+ appear outside per net 2e- transferred in 2 cycles, only 2H+ are taken up on the matrix side. In respiratory chain complex IV (see below), there is a similarly uncompensated uptake of protons from the matrix side (4H+ per O2 or 2 per 2e-). Thus there are 2H+ per 2e- that are effectively transported by a combination of complexes III & IV. They are listed with complex III in diagrams (e.g., see above) depicting H+/e- stoichiometry.
of e- & H+ transfer in Complex III Half of the homo-dimeric complex III is depicted at right. The approximate location of the membrane bilayer is indicated. Not shown are the CoQ binding sites near heme bH near heme bL.
The b hemes are positioned to provide a pathway for electron transfer across the membrane.
The protein domain with attached Rieske iron-sulfur center (labeled Fe-S) has a flexible link to the rest of the complex. At right, the iron-sulfur center protein is colored green. The iron-sulfur center changes position during electron transfer. After Fe-S extracts an e- from QH2, it moves closer to heme c1 (cytochrome c1) to which it transfers the e-. View an animation of this domain movement by the Crofts lab. After the first electron transfer from QH2 to Fe-S, the CoQ semiquinone is postulated to shift position within the Q-binding site, moving closer to its electron acceptor, heme bL. This would help to prevent transfer of the second electron from the semiquinone to Fe-S. Complex III is an obligate homo-dimer. The iron-sulfur center in one half of the dimer may interact with bound CoQ and heme c1 in the other half of the dimer.
At right, arrows indicate the positions of: Fe-S in the half of the complex whose proteins are colored white/grey heme c1 in the half of the complex whose proteins are colored in shades of blue or green.
Crystal structures on which these diagrams are based, (PDB 1BE3 & 1BGY) were solved by S. Iwata et al, in 1998.
Complex IV (Cytochrome Oxidase): As discussed in the section on the respiratory chain, electrons are donated to complex IV, one at a time, by cytochrome c, which binds from the intermembrane space. Each electron passes via CuA and heme a to the binuclear center, buried within the complex, that catalyzes oxygen reduction: 4e- + 4H+ + O2 > 2H2O. Protons utilized in this reaction are taken up from the matrix compartment.
H+ pumping by complex IV: In addition to the protons utilized in the reduction of O2, there is electron transfer-linked transport of 2H+ per 2e- (4H+ per 4e-) from the matrix to the intermembrane space.
Structural and mutational studies indicate that protons pass through complex IV via chains of groups subject to protonation/deprotonation, called "proton wires." These consist mainly of chains of buried water molecules, along with amino acid side-chains, and propionate side-chains of the hemes.
Separate H+-conducting pathways link each side of the membrane to the buried binuclear center where O2 reduction takes place. These include two proton pathways, designated "D" and "K" (named after constituent Asp and Lys residues) extending from the mitochondrial matrix to near the binuclear center deep within complex IV. See diagram p. 826, and images in: a webpage maintained by the Institute of Biological Information Processing in Germany, a webpage maintained by A. Crofts.
A switch mechanism controlled by the reaction cycle is proposed to effect transfer of a proton from one half-wire (half-channel) to the other. There cannot be an open pathway for H+ completely through the membrane, or oxidative phosphorylation would be uncoupled. (Pumped protons would leak back; see below). The process of switching may involve conformational changes, and oxidation/reduction-linked changes in pKa of groups associated with the catalytic metal centers. Detailed mechanisms have been proposed (see articles on oxidase). A simplified animation of the entire respiratory chain, at right, includes a simplified representation of the Q cycle. A total of 20 H+ is shown being transported out of the matrix, per 4 e- transferred from 2 NADH to O2 (10 H+ per ? O2).
Not shown are OH- ions that would accumulate in the matrix, as protons, generated by dissociation of water (H2O ?a H+ + OH-), are pumped out. Also not depicted is the effect on the pH gradient of buffering.
of the Respiratory Chain
The ATP synthase, which is embedded in cristae of the inner mitochondrial membrane, includes the following major subunits: F1 - the catalytic subunit, made of 5 polypeptides with stoichiometry a3b3gde. Fo - a complex of integral membrane proteins that mediates proton transport. The F1Fo complex couples ATP synthesis to H+ transport into the mitochondrial matrix. Transport of at least 3H+ per ATP synthesized is required, as estimated from a comparison of the following (calculated in the Tutorial):
the free energy change (DG) associated with synthesis of ATP under cellular conditions (the free energy required)
the free energy change (DG) associated with transport of each H+ into the mitochondrial matrix, based on the electrochemical H+ gradient (the free energy available per H+).
ATP Synthase structure and mechanism are discussed in more detail elsewhere.
The Chemiosmotic Theory of oxidative phosphorylation, for which Peter Mitchell received the Nobel prize is summarized in the diagram at right.
The Chemiosmotic Theory states that coupling of electron transfer to ATP synthesis is indirect, via a H+ electrochemical gradient:
Respiration: Spontaneous electron transfer through complexes I, III, and IV is coupled to non-spontaneous H+ ejection from the mitochondrial matrix. H+ ejection creates a membrane potential (DY, negative in the matrix) and a pH gradient (DpH, alkaline in the matrix).
F1Fo ATP Synthase: Non-spontaneous ATP synthesis is coupled to spontaneous H+ transport into the matrix compartment. The pH and electrical gradients created by respiration are together the driving force for H+ uptake. Return of protons to the matrix via Fo "uses up" the pH and electrical gradients.
ATP produced in the mitochondria must exit to the cytosol to be used by transport pumps, kinases, etc. ADP and Pi, arising from ATP hydrolysis in the cytosol, must re-enter the mitochondria to be converted again to ATP.
Two carrier proteins in the inner mitochondrial membrane are required for this metabolic cycle. The outer membrane is considered to be not a permeability barrier. The large VDAC channels in the outer membrane are assumed to allow passage of adenine nucleotides and Pi.
The Adenine Nucleotide Translocase (ADP/ATP carrier) is an antiporter that catalyzes exchange of ADP for ATP across the inner mitochondrial membrane (p. 496). At cellular pH, ATP has four negative charges, while ADP has 3 negative charges. ADP3-/ATP4- exchange is driven by, and uses up, the membrane potential generated by respiration (one charge per ATP)
Peter Mitchell was born in Mitcham, in the County of Surrey, England, on September 29, 1920. His parents, Christopher Gibbs Mitchell and Kate Beatrice Dorothy (nee) Taplin, were very different from each other temperamentally. His mother was a shy and gentle person of very independent thought and action, with strong artistic perceptiveness. Being a rationalist and an atheist, she taught him that he must accept responsibility for his own destiny, and especially for his failings in life. That early influence may well have led him to adopt the religious atheistic personal philosophy to which he has adhered since the age of about fifteen. His father was a much more conventional person than his mother, and was awarded the O.B.E. for his success as a Civil Servant.
Peter Mitchell was educated at Queens College, Taunton, and at Jesus college, Cambridge. At Queens he benefited particularly from the influence of the Headmaster, C.L. Wiseman, who was an excellent mathematics teacher and an accomplished amateur musician. The result of the scholarship examination that he took to enter Jesus College Cambridge was so dismally bad that he was only admitted to the University at all on the strength of a personal letter written by C.L. Wiseman. He entered Jesus College just after the commencement of war with Germany in 1939. In Part I of the Natural Sciences Tripos he studied physics, chemistry, physiology, mathematics and biochemistry, and obtained a Class III result. In part II, he studied biochemistry, and obtained a II-I result for his Honours Degree.
He accepted a research post in the Department of Biochemistry, Cambridge, in 1942 at the invitation of J.F. Danielli. He was very fortunate to be Danielli's only Ph.D. student at that time, and greatly enjoyed and benefited from Danielli's friendly and unauthoritarian style of research supervision. Danielli introduced him to David Keilin, whom he came to love and respect more than any other scientist of his acquaintance.
He received the degree of Ph.D. in early 1951 for work on the mode of action of penicillin, and held the post of Demonstrator at the Department of Biochemistry, Cambridge, from 1950 to 1955. In 1955 he was invited by Professor Michael Swann to set up and direct a biochemical research unit, called the Chemical Biology Unit, in the Department of Zoology, Edinburgh University, where he was appointed to a Senior Lectureship in 1961, to a Readership in 1962, and where he remained until acute gastric ulcers led to his resignation after a period of leave in 1963.
From 1963 to 1965, he withdrew completely from scientific research, and acted as architect and master of works, directly supervising the restoration of an attractive Regency-fronted Mansion, known as Glynn House, in the beautiful wooded Glynn Valley, near Bodmin, Cornwall - adapting and furnishing a major part of it for use as a research labotatory. In this, he was lucky to receive the enthusiastic support of his former research colleague Jennifer Moyle. He and Jennifer Moyle founded a charitable company, known as Glynn Research Ltd., to promote fundamental biological research and finance the work of the Glynn Research Laboratories at Glynn House. The original endowment of about ?250,000 was donated about equally by Peter Mitchell and his elder brother Christopher John Mitchell.
In 1965, Peter Mitchell and Jennifer Moyle, with the practical help of one technician, Roy Mitchell (unrelated to Peter Mitchell), and with the administrative help of their company secretary, embarked on the programme of research on chemiosmotic reactions and reaction systems for which the Glynn Research Institute has become known. Since its inception, the Glynn Research Institute has not had sufficient financial resources to employ more than three research workers, including the Research Director, on its permanent staff. He has continued to act as Director of Research at the Glynn Research Institute up to the present time. An acute lack of funds has recently led to the possibility that the Glynn Research Institute may have to close.
Beside his interest in communication between molecules, Peter Mitchell has become more and more interested in the problems of communication between individual people in civilised societies, especially in the context of the spread of violence in the increasingly collectivist societies in most parts of the world. His own experience of small and large organisations in the scientific world has led him to regard the small organisations as being, not only more alive and congenial, but also more effective, for many (although perhaps not all) purposes. He would therefore like to have the opportunity to become more deeply involved in studies of the ways in which sympathetic communication and cooperative activity between free and potentially independent people may be improved. One of his specific interests in this field of knowledge is the use of money as an instrument of personal responsibility and of choice in free societies, and the flagrant abuse and basically dishonest manipulation of the system of monetary units of value practised by the governments of most nations.
|
|
| free web hits counter |
This is my BrainyGoose:
United States, IL, Chicago, English, Italian, Genry, Male, 21-25, bodybulding, swiming.