Animal respiration has been of serious interest to scientists since 1777, when Lavoisier concluded that foods undergo slow combustion within the body, supposedly in the blood. In 1803–1807, Spallanzani established for the first time that the tissues were the actual site of respiration, but his conclusions were largely ignored. In 1884, MacMunn discovered that cells contain the heme pigments, which are now known as cytochromes. However, the leading biochemists of the day dismissed the observations as experimental error, and it was not until the present century that serious study of the chemistry of bio- logical oxidations began.a,b
Recognition that substrates are oxidized by dehydrogenation is usually attributed to H. Wieland. During the years 1912–1922 he showed that synthetic dyes, such as methylene blue, could be substituted for oxygen and would allow res- piration of cells in the absence of O2. Subsequent experiments (see Chapter 15) led to isolation of the soluble pyridine nucleotides and flavoproteins and to development of the concept of an electron trans- port chain.
Looking at the other end of the respiratory chain, Otto Warburgc,d noted in 1908 that all aerobic
cells contain iron. Moreover, iron-containing char- coal prepared from blood catalyzed nonenzymatic oxidation of many substances, but iron-free charcoal prepared from cane sugar did not. Cyanide was found to inhibit tissue respiration at low concentra- tions similar to those needed to inhibit nonenzymat- ic catalysis by iron salts. On the basis of these investigations, Warburg proposed in 1925 that aerobic cells contain an iron-based Atmungsferment (respira- tion enzyme), which was later called cytochrome oxidase. It was inhibited by carbon monoxide.
Knowing that carbon monoxide complexes of hemes are dissociated by light, Warburg and Negelein, in 1928, determined the photochemical action spectrum (see Chapter 23) for reversal of the carbon monoxide inhibition of respiration of the yeast
Torula utilis. The spectrum closely resembled the
absorption spectrum of known heme derivatives (Fig. 16-7). Thus, it was proposed that O2, as well as CO, combines with the iron of the heme group in the Atmungsferment.
Meanwhile, during 1919–1925, David Keilin, while peering through a microscope equipped with a spectroscopic ocular at thoracic muscles of flies and other insects, observed a pigment with four distinct absorption bands. At first he thought it was derived by some modification of hemoglobin, but when he found the same pigment in fresh baker’s yeast, he recognized it as an important new
substance. In his words:e
One day while I was examing a suspension of yeast freshly prepared from a few bits of compressed yeast shaken vigorously with a little water in a test-tube, I failed to find the characteristic four-banded absorption spectrum, but before I had time to remove the suspension from the field of vision of the microspectroscope the four absorption bands suddenly reappeared. This experiment was repeated time after time and always with the same result: the absorption bands disappeared on shaking the suspension with air and reappeared within a few seconds on standing.
I must admit that this first visual perception of an intracellular respiratory process was one of the most impressive spectacles I have witnessed in the course of my work. Now I have no doubt that cytochrome is not only widely distributed in nature and completely inde- pendent of haemoglobin, but that it is an intracellular respiratory pigment which is much more important than haemoglobin.
Keilin soon realized that three of the absorption bands, those at 604, 564, and 550 nm (a, b, and c), represented different pigments, while the one at 521 nm was common to all three. Keilin proposed the names cytochromes a, b, and c. The idea of an elec- tron transport or respiratory chain followede quickly
as the flavin and pyridine nucleotide coenzymes were recognized to play their role at the dehydro- genase level. Hydrogen removed from substrates by these carriers could be used to oxidize reduced cytochromes. The latter would be oxidized by oxygen under the influence of cytochrome oxidase.
In 1929, Fiske and Subbarow,d,f–h curious about
the occurrence of purine compounds in muscle extracts, discovered and characterized ATP. It was soon shown (largely through the work of Lundsgaard and Lohman)f that hydrolysis of ATP
provided energy for muscular contraction. At about the same time, it was learned that synthesis of ATP accompanied glycolysis. That ATP could also be formed as a result of electron transport became clear following an observation of Engelhardth,i in 1930,
that methylene blue stimulated ATP synthesis by tissues.
The study of electron transport chains and of oxidative phosphorylation began in earnest after Kennedy and Lehninger,j in 1949, showed that
mitochondria were the site not only of ATP synthe- sis but also of the operation of the citric acid cycle and fatty acid oxidation pathways. By 1959, Chance had introduced elegant new techniques of spectro- photometry that led to formulation of the electron
BOX 18-A (continued)
transport chain as follows:
Substrate→ pyridine nucleotides → flavoprotein → cyt b→ cyt c → cyt a → cyt a3→ O2
Since that time, some new components have been added, notably the ubiquinones and iron- sulfur proteins, but the basic form proposed for the chain was correct.
a Kalckar, H. M. (1969) Biological Phosphorylations, Prentice-Hall,
Englewood Cliffs, New Jersey
b Kalckar, H. M. (1991) Ann. Rev. Biochem. 60, 1 – 37
c Edsall, J. T. (1979) Science 205, 384 – 385
d Fiske, C. H., and Subbarow, Y. (1929) Science 70, 381 – 382
e Keilin, D. (1966) The History of Cell Respiration and Cytochrome,
Cambridge Univ. Press, London and New York
f Kalckar, H. (1980) Trends Biochem. Sci. 5, 56 – 57
g Schlenk, F. (1987) Trends Biochem. Sci. 12, 367 – 368
h Saraste, M. (1998) Science 283, 1488 – 1493
i Slater, E. C. (1981) Trends Biochem. Sci. 6, 226 – 227
j Talalay, P., and Lane, M. D. (1986) Trends Biochem. Sci. 11, 356–358
membrane bilayer. Membranes also contain ubiqui- none-binding proteins,66,67 which probably hold the
ubiquinone that is actively involved in electron trans- port. Perhaps some ubiquinone molecules function as fixed carriers. There is also uncertainty about the number of sites at which ubiquinone functions in the chain.
Mitochondrial electron transport in plants and fungi. Plant mitochondria resemble those of mammals in many ways, but they contain additional dehydro- genases and sometimes utilize alternative pathways of electron transport,68– 73 as do fungi.74 Mitochondria are
impermeable to NADH and NAD+. Animal mitochon-
dria have shuttle systems (see Fig. 18-16) for bringing the reducing equivalents of NADH into mitochondria
and to the NADH dehydrogenase that faces the matrix side of the inner membrane. However, plant mito- chondria also have an NADH dehydrogenase on the outer surface of the inner membrane (Fig. 18-6). This enzyme transfers electrons to ubiquinone, is not inhibited by rotenone (see Fig. 18-5), and also acts on NADPH. Inside the mitochondria a high-affinity NADH dehydrogenase resembles complex I of animal mitochondria and is inhibited by rotenone.73 There is
also a low-affinity NADH dehydrogenase, which is insensitive to rotenone. Some plant mitochondria respire slowly in the presence of cyanide. They utilize an alternative oxidase that replaces complex III and cytochome c oxidase and which is not inhibited by antimycin or by cyanide (Fig. 18-6).68,71,75 It is especially
active in thermogenic plant tissues (Box 18-C). A
D I III IV Q QH2 Succinate Fumarate b FeS c NADH NAD+ NADH NAD+ Glycerol-P Dihydroxy- acetone-P NAD(P)H NAD(P)+ O2 H2O O2 H 2O Matrix C B c1 Cytosol (Intermembrane) II A
Figure 18-6 Schematic diagram of some mitochondrial dehydrogenase and oxidase complexes of plants and also the glycerol
phosphate dehydrogenase of animals, which is embedded in the inner membrane. Complexes I–IV are also shown. (A) The glycerol phosphate dehydrogenase of some animal tissues. It is accessible from the intermembrane space on the cytosolic side. (B) The rotenone-insensitive NAD(P)H dehydrogenase of the external membrane surface of plants. (C) The rotenone- insensitive NADH dehydrogenase facing the matrix side in some plants. (D) The plant alternative oxidase. Ubiquinone, Q.