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1.History

Mitochondria. Immo E. Scheffler

mitochondria as the “powerhouse of the cell.” Some of the most illustrious names in the early history of biochemistry can be found among contributors to the evolution of this concept. The discoveries of cytochromes, iron-porphyrin compounds (heme), flavin and pyridine nucleotides, and various dye-reducing dehydrogenases fall into the period between 1920 and the 1930s, and the formulation of the citric acid cycle by Krebs was one of the crowning achievements of the study of metabolism and respi-ration in muscle preparations.

Although K. Lohmann discovered adenosine triphosphate (ATP) in 1931, it took ten more years to demonstrate its general role beyond muscle, and during this period O. Warburg and O. Meyerhof described what is now referred to as substrate level phosphorylation (ATP synthesis coupled to the enzymatic oxidation of compounds such as glyceraldehyde phosphate), in contrast to oxidative phosphorylation, first shown by H.M. Kalckar to be firmly coupled to respiration (1937–1941).

This was also a time when biochemists proceeded to grind up tissue, filter it through cheesecloth, perhaps even centrifuge the mixture at some indeterminate speed, and then threw away everything but the supernatant. Insoluble particles con-stituted a nuisance and an insurmountable obstacle in the purification of an enzyme, and students were urged not to “waste clean thoughts on impure enzymes.” The study of mitochondria required the isolation and purification of larger quantities of the or-ganelle, and the first attempts in this direction were doomed by the use of unsuitable buffers and media for cell suspension and breakage.

Cell Biology metamorphosed out of the older science of Cytology when two pow-erful new methodologies were perfected and applied to the study of biological tissues. Pioneering advances and applications were made in parallel and synergistically at the same institution during the 1940s: at the Rockefeller Institute, A. Claude and his col-leagues began to use the centrifuge as a sophisticated analytical tool for the fraction-ation of subcellular structures (differential centrifugation), a technique which C. DeDuve [2] would later describe as “exploring cells with a centrifuge.” Subcellular particles were fractionated reproducibly and with increasing resolution to achieve pure fractions. At the same time, careful biochemical characterizations were carried out. Important concepts to emerge were the recognition of the polydisperse popula-tion of particles (size variation), and the postulate of biochemical homogeneity. Mi-crosomes and mitochondria were represented by overlapping populations of granules of different size, but at the large end were almost pure mitochondria, and at the small end were almost pure microsomes (which were later resolved further into lysosomes and peroxisomes [2]). In 1948 G.H. Hogeboom, W.C. Schneider, and G. Palade dis-covered that 0.88 M sucrose greatly stabilized mitochondria, which aided their isola-tion from liver in a morphologically intact form.

The criterion of morphological intactness was practicable only because the groups led by K. Porter and G. Palade used the electron microscope in the exploration of cells. Particles could now be viewed and compared in situ and after isolation by dif-ferential centrifugation, so that investigators felt confident that the particle was intact, and therefore most likely completely functional.

Nevertheless, isolating and looking at a particle does not immediately give many clues about its biological function, although remarkably prescient guesses and de-

ductions had been made from staining experiments. An approach from a different di-rection finally led to the full appreciation of the role of mitochondria in respiration.

A.L. Lehninger (and independently L.F. Leloir and J.M. Munoz) in the period 1943–1947 had focused on the oxidation of fatty acids in liver homogenates and found the activity to be dependent on an insoluble component that was sensitive to os-motic conditions. The newly established conditions for mitochondrial isolation were applied by E. Kennedy and A.L. Lehninger to prove that fatty acid oxidase activity of the liver was found almost exclusively in mitochondria. The same investigators then extended the biochemical characterization of these organelles by demonstrating that 1) the reactions of the citric acid cycle can be carried out in mitochondria at a rate that can account for most if not all of the activity found in liver cells, and 2) such reactions were accompanied by the synthesis of ATP (oxidative phosphorylation).

Just as a cell was recognized to be much more than a “bag of enzymes,” it soon be-came clear that many of the enzymes catalyzing the biochemical reactions observed in mitochondria were not simply contained within this organelle in soluble form by the mitochondrial membrane. In fact, the potential complexity of this organelle be-came apparent from the early electron microscopic observations which revealed the existence of an inner and an outer membrane, with the inner membrane often highly folded (termed cristae by G. Palade). Topologically one can therefore distinguish two spaces inside the mitochondrion: the intermembrane space and the matrix. However, a full appreciation of the significance of this compartmentalization was probably not achieved until much later.

The enzymes for fatty acid oxidation and for the citric acid cycle (with the excep-tion of SDH) were found to be soluble in the mitochondrial matrix. The enzymes re-sponsible for oxidation of NADH and electron transport to oxygen were insoluble and localized to the inner membrane (cristae). A more detailed description of their char-acterization is given in Chapters 5 and 6. Here we mention two accomplishments of the 1960s. First, the overall arrangement of the components of the electron transport chain and the flow of electrons from dehydrogenases to flavoproteins to various non-heme iron-sulfur centers and cytochromes and finally to oxygen, first glimpsed by D. Keilin in the 1930s, was established by a combination of spectroscopic studies and the use of specific inhibitors such as rotenone, antimycin, and cyanide in various labora-tories. The studies of B. Chance deserve special mention. D.E. Green was another in-fluential investigator in the 1950s and 1960s. While his ideas about an “elementary particle” within mitochondria have not stood up to the test of time, his Institute for Enzyme Research was the training ground for a number of prominent researchers in the subsequent decades. A very informative, entertaining, and highly personalized ac-count has been written by one of the pioneers in the field, E. Racker [3]. Second, the efforts of Y. Hatefi and colleagues [4,5] culminated in the fractionation and charac-terization of five multisubunit complexes from the inner membrane: four are involved in the respiratory chain, and the fifth was identified as the site of the phosphoryla-tion of ADP to ATP. Among the current high points in the biochemical and structural analysis of these complexes is undoubtedly the recent achievement of high-resolution structures for complex III [6], complex IV (cytochrome oxidase) [7] and the F

com-

1 ponent of complex V (ATP synthase) [8–10] by x-ray diffraction and other biophysi-

cal means. A Nobel Prize in Chemistry has just been awarded for the elucidation of the structure and mechanism of the F

–ATP synthase to J. Walker of Cambridge Uni-

versity and P. Boyer at the University of California at Los Angeles.

The distinction between substrate level phosphorylation and oxidative phosphory-lation is the subject of examination questions for thousands of undergraduate students every year. The former is straightforward to explain in terms of enzyme kinetics and the coupling of exergonic and endergonic enzyme-catalyzed reactions. The challenge to explain oxidative phosphorylation has preoccupied some of the best biochemists for a good part of their career. An ingenious solution, offered by P. Mitchell [11], was slow to be accepted, but it eventually revolutionized our thinking about bioenergetics, membranes, membrane potentials, active transport, ion pumps, and “vectorial metab-olism.” A detailed understanding of the structure of membranes, and the relationship between lipids and integral membrane proteins, was a prerequisite [12]. The chemios-motic hypothesis has found universal acceptance not only in explaining oxidative phosphorylation in mitochondria, but also aspects of photosynthesis in chloroplasts, and light-driven phosphorylations in bacteria.

While failing to live up to early expectations of mitochondria as the carriers of all hereditary information, a most important discovery was made in 1963 when one of the first definite identifications of DNA in mitochondria was made [13]. This dis-covery had in many ways been anticipated by the discovery of nonmendelian, cyto-plasmic inheritance in yeast by the Ephrussi laboratory [14]. Ramifications of this discovery are wide. It renewed and strengthened interest in the evolutionary origin of mitochondria. The problem of understanding how two genomes, nuclear and mito-chondrial genes, interact in the biogenesis of this organelle is still an acute one. In ver-tebrates the mitochondrial genome is relatively small, deduced from isolated circular mtDNA molecules visualized by electron microscopy. The complete sequencing of a mammalian mtDNA in Cambridge was one of the earliest triumphs of the DNA se-quencing technology invented by F. Sanger and his colleagues in the 1970s [15]. Al-most simultaneously, G. Attardi [16] and his coworkers at the California Institute of Technology determined the nature of the transcripts derived from this genome and set the stage for the identification of all the genes encoded by mammalian mtDNA. Changes in mitochondrial DNA sequences are believed to represent a molecular clock on a time scale that appears particularly relevant for human evolution, and provoca-tive ideas and speculations have centered around deductions from such sequence comparisons, with implications for primate and human evolution (“the Mitochondrial Eve”), and the spread of human populations by global migrations. Controversies still center around the question whether this clock is more accurate than a sundial (e.g., N. Howell). Currently, forensic investigations utilize information about mitochondrial DNA sequences from victims and suspects. Finally, where there is DNA, there must be mutations. An explosion of publications in the last decade, triggered by the pio-neering work of Wallace and his colleagues [17] and Holt and colleagues [18], has de-scribed mutations in mitochondrial DNA that are directly responsible for human genetic diseases (myopathies and neuropathies), while speculations go even further in relating accumulating defects in mitochondrial DNA to a variety of ailments accom-panying aging and senescence.

REFERENCES5 Thus, mitochondria occupy a central position in our understanding of the cell, the “basic unit of life,” and the study of mitochondria has, from the very beginning, re-vealed not only details, but also fundamental insights covering the entire spectrum from biophysics to cell biology and genetics. Their demonstrated role in disease, and their implied role in neurodegenerative developments in advanced age makes them particularly fashionable now, but a history of biochemistry is unthinkable without ref-erence to mitochondria and the monumental discoveries made in the course of their study. Similarly, the characterization of mitochondrial morphology by electron mi-croscopy in intact cells and tissues and after cell fractionation (e.g., by G. Palade and F.S. Sjostrand and coworkers) coincided with and even triggered the emergence of Cell Biology as a distinct science. The discovery of these and other organelles em-phasized the compartmentalization of the cytosol, and hence forced investigators to focus on exploring the various mechanisms of protein targeting to distinct organelles. Today, one of several frontiers is the integration of mitochondria into the cell and their distribution in the cytosol by means of their interaction with the cytoskeleton, espe-cially in various highly differentiated cells. Many questions remain, and an under-standing of the achievements described in the following chapters will help to define future directions.

REFERENCES

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*3.Racker, E. (1976) A New Look at Mechanisms in Bioenergetics, Academic Press, New Y ork 4.Hatefi, Y., Galante, Y.M., Stiggal, D.L., and Ragan, C.I. (1979) Methods Enzymol.56,

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6HISTOR Y

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