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The discovery of cytochrome
BIOCHEMICAL EDUCATION
October 1973 Vol. 1 No. 4
THE DISCOVERY OF CYTOCHROME
E.F. HARTREE A.R.C. Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road,
69
i
In these days, when the world of science is under pressure to organize for the pursuit of practical ends, when the scale of scientific endeavour is making the lone furrow an anachronism, it is salutory to recall the single-hande0, achievements of men of science stimulated solely by an urge ~o.understand the living world. The discovery of cytochrome t l ) is a notable example of such achievements not only because of the disarming simplicity of experimental approach by the discoverer, David Kellin, but also because the discovery came at a critical moment. It resolved a serious dilemma that was impeding the evolution of biochemistry from an untidy and rather primitive branch of chemistry into a major scientific discipline: mote succinctly the tranfformation of bio-Chemistry into Biochemistry. Keilin's early life was spent in Warsaw but in 1904, at the age of 17, he began his university career in Li[ge, moving the following year to Paris. One day he happened to take shelter from the rain in a building where a lecture was in progress. This was the beginning of a life-long friendship with the lecturer, Manrice Caullery, and of Keilin's career in biology. At the Sorbonne he read geology, botany, zoology and embryology, while simultaneously carrying out research under Cauilery in parasitology and entomology. By 1915, when he had achieved high reputation as a parasitologist, he came to Cambridge to join George Nuttall (Quick Professor of Biology) and to continue his research on parasitic Diptera. In 1921 Nuttall's group moved t o the newly-built Molteno Institute with which Keilin's name is inseparably linked. At this time Keilin was deeply interested in the striking evolutionary adaptations of dipterous larvae. Many such adaptations are the consequence of a larva's food being situated in a medium deficient in oxygen. Am9ng objects of study were larvae of the fly Gasterophilus intestinalis tl)which parasitize the stomachs of horses, becoming attached to the mucosa by mouth-hooks. The immature larvae are uniformly red but as they grow the pigment becomes localized in trachael ceils which fill the posterior third of the body. These cells, forming integral parts of the larva's respiratory system, each contain an extensive ramification of fine tracheoles which branch off from the trachae. In order to characterize the pigment Keilin used a microspectroscope which is a direct-vision spectroscope ocular designed to replace the eygpiece of a microscope. He placed a living larva above the microscope condenser lens, compressed it lightly with a microscope slide, and illuminated it with a high-intensity light source. The red transmitted light revealed in the microspectroscope the two characteristic absorption bands of oxyhaemoglobin. These bands were however at wavelengths slightly different from those of horse oxyhaemogiobin. The pigment is thus a ~ecific Gasterophllus oxyhaemoglobin. Noting that no tissues of the adult fly are red Keilin was curious about the fate of this oxyhaemogiobin. He therefore examined spectroscopically tissues dissected from the adult. He found that the wing-muscles, which were pale orange-pink by transmitted light, showed four sharp absorption bands. None of these bands corresponded, in terms of wavelength, to the bands of oxyhaemogiobin. He then examined tissues of blow-flies and other insects and without exception he detected in the wing-muscles the same four-banded spectrum. Having cultures of Bacillus subtilis on hand Keilin was inspired to examine them spectroscopically. He scraped the growing cells from agar plates and placed the cheesy mass under the microscope as a thin layer on a glass slide. Again the same four-banded spectrum! This result led him to examine a flattened pellet of baker's yeast - and once more with the same result. It was now clear that the new pigment was not a transformation product of oxyhaemoglobin.
Cambridge CB30JQ, England. Wishing to study the effects of reagents upon the pigment Keilin prepared a thick suspension of yeast by shaking it vigorously with water in a test-tube. Surprisingly, an immediate examination of the suspension revealed only diffuse and scarcely perceptible absorption bands. But after a few seconds' delay the four sharp bands appeared quite suddenly. This remarkable phenomenon could be demonstrated repeatedly with the same sample of yeast suspension: if it was examined immediately after vigorous shaking the bands could not be detected but within 5 - 1 0 seconds, while the spectrum was kept under observation, the four bands reappeared. Keilin's interpretation of these experiments was as follows:- The yeast pigment, to which • he gave the name cytochrome, became oxidised during the shaking in air and in the oxidised form it showed no sharp absorption bands. When shaking was interrupted the respiratory activity of the yeast led to a progressive decrease in the concentration of dissolved oxygen and to reduction of cytochrome, the reduced form of which shows the four-banded spectrum. In other words cytochrome was acting as a respiratory carrier linking oxygen with the oxidizable metabolites in the living yeast cell. Substantial support for this hypothesis soon followed. Thus respiratory poisons (substances that reduce the rate of oxygen consumption by aerated yeast suspensions) also I~locked the spectroscopicaily observed oxidation-reduction cycle of cytochrome. For example, cyanide prevented the oxidation of cytochrome by aeration while urethane greatly delayed the reduction of cytochrome after aeration had ceased. The rate of reduction was also slow in starved yeast, i.e. yeast which, as an aqueous suspension, had been aerated for a long
DAVID KEILIN 1887 - 1963
70
BIOCHEMICAL EDUCATION
period to exhaust stored metabolites. Further study showed that cytochrome was very widely distributed, being present in many animal and plant tissues as well as in yeasts and bacteria. This survey yielded two noteworthy results. Firstly, the cytochrome spectrum showed a remarkably constant pattern: reading from the red end of the spectrum Keilin designated the bands a, b, c, and d. The first three were sharp and prominent while d was fainter and more diffuse as though consisting of several weaker, but clostly situated bands. In bakers' yeast the wavelengths of the band centres were: a, 604 nm; b, 564 nm; c, 550 nm; d, 521 nm. In most other tissues the wavelengths varied only slightly from the yeast values, although wider variations in pattern were found among aerobic bacteria. Several strictly anaerobic bacteria contained no detectable cytochrome. Secondly, there was an obvious relationship between the intensity of absorption bands (i.e. the concentration of cytochrome) in a tissue and the respiratory activity of that tissue. A high cytochrome content was noted in yeast, aerobic bacteria, flight muscles of insects and birds, heart-muscle and brain grey matter of mammals. Detection of cytochrome in mammalian tissue was facilitated if blood was removed by perfusion. Study of two moths provided results of considerabl.e interest. The male winter-moth, Cheimotobia brumata, flies and has well-developed thoracic muscles which are rich in cytochrome. The female" has rudimentary, non-functional wings and the musculature shows a very feeble cytochrome spectrum. However, it was with the wax-moth, Galleria mellonella, that Keflin carried out his most fascinating experiments on the functioning of cytochrome in a living organism. He attached a living specimen to a glass slide with gum arabic, illuminated the thorax from below and studied the transmitted light with the microspectroscope. Periodically the moth would attempt to detach itself by vibrating the wings. While this was happening strong cytochrome bands could be seen. During intervening quiescent periods the absorption bands were very weak. When the immobilized insect was placed in a glass box and a trace of hydrogen cyanide was introduced the insect remained immobile but strong cytochrome bands appeared. Replacing air in the box by nitrogen also caused cytochrome bands to appear. In both cases flushing the box with air led to a return of periodic wing vibration and to the previously observed oxidation and reduction of cytochrome. In discussing his experiments Keilin emphasized that the state of cytochrome in living tissues is a dynamic equilibrium between two reactions, oxidation and reduction, both of which are proceeding rapidly. Thus the reduction of cytochrome in presence of cyanide is not d n a t o any reducing action of cyanide but to an interference with the oxidation process and the establishment of a new equilibrium. Because of its widespread occurrence Keilin scarcely believed that cytochtome, could have remained unnoticed by others. After much searching tl ? he found in old text-books of physiology confusing references to "myohaematin", a pigment which was reputed to occur in muscle and other animal tissues. Following these leads Keilin came a c r ~ papers published in 1884-1887 by C.A. MacMunn ~ " , a medical practitioner and, in his spare time, a spectrolcopist. MacMunn had given the names myohaematin and histohsmatin to pigments characterized by four-banded absorption
*In Ma~lunn's time the term haemochromogen was applied in~inately to reddish breakdown products of haemoglobin that showed prominent absorption bands in the visible spectrum. By 1925 the term was commonly applied to denatmed haemoginbin. Native haemoglobin is a complex of a specific protein, globin, with ferrous protoporphyrin (commonly known as heem). When haemoglobin is denatured, e.g. by heat or alkali, and a r e d U ~ agent is added the remlting complex of h u m and , denatured globin is designated 81obin haemochromogen. Haem freed fro m globin also forms haemochromogens with other denatured proteins. Reducing conditions are essential since haem reacts readily with oxygen to give haematin (i.e. ferric protoporphyrin). The spectrum of haemochromogen consists of two sharp bands of unequal intensity: a strong or-band and, nearer to
October 1973 Vol. 1 No. 4
spectra that he detected in a wide range of animal tismes from sponges to man. He obtained spectra of the oxidized and reduced forms by chemical means (addition of hydrogen peroxide and ammonium sulphide resl~ctively). Although he did not observe rever~ble oxidation and reduction in living tissues he was convinced that the pigments were respiratory carriers. His papers came under attack from some of the "big guns" of physiology who concluded that MacMunn's four-banded spectrum represented no more than a mixture of known haematin pigments (heemoproteins) such as myoglobin and haemoglobin together with haemochromogen derived from these pigments. Such criticilans were accepted by MacMunn's contemporaries and his work was forgotten. Because of his interest in Gasterophilus Keilin had familim~ed himself with the properties, in particular the spectroscopy, of derivatives of haematin: e.g. the oxygenated and deoxygenated forms of haemoglobin arld myoglobin as well as h a e m o d l r o ~ s and related" compounds. Being able to distinguish mixtures of sttch compounds from cytochrome Kenin was not only able to grasp the significance of MacMunn's work but also to unravel the confused and sometimes unwarranted criticisms to which this work had been subjected.* Keilin noticed that when a layer of yeast was left to dry and was examined periodically with the microspectroscope the a-band disappeared first. The b-band d ~ a r e d late~ alid finally the c- and d-bands faded simultaneouS. Extraction of fresh yeast with hot water or dried yeast with cold water yielded soltttions showing band c and a narrowed band d. KeRin concluded that c y t ~ o m e consists of three component pigments, which he designated cytochromes a, b and c, each showing a haemochromogen-type spectrum in the reduced form. t Thus bands a, b, and c are the three strong fl,hands of the three components and band d represents fused weak ~-bands.The components were regarded as "native haemochromollens" since they did not combine with carbon monoxide and were not readily autoxidizable, i.e. they did not become oxidized if shaken with air in presence of a trace of cyanide (the role of cyanide is to inhibit the enzyme cytochrome oxidase - see below). When cytochtome was denatured the absorption bands shifted slightly and the components acquired normal properties of heemochromogens: they became autooxidizable in presenCe of cyanide and they formed spectroscopically recognizable carbon monoxide derivatives. At this stage of his work Keilln published hisJ~i~st paper on cytochrome in the Proceedings of the Royal Society t3.,. The same year saw the beginning of a remarkable period in Keilin's careez. It was in that year that he was appointed university lecturer in parasitology. This involved him in a heavy programme of lectures and practical classes. Within the period 1925-31 he reached the zenith of his career as an experimental parasitologist and published ten papers on the subject. His spare time he devoted to cytochrome, publishing nine papers in this field in the years up to 1931, He was sole author of the first six (3, 4) and R was these that established cytochrome as the keystone of the respiratory mechanism in aerobic cells. Although he subsequently gave up parasitological research he continued to
the blue end of the spectrum, a weak ~-hand. The two bands of oxyhaemogiobin are of nearly equal intensity and are at wavelengths different from those of the derived heemochromogen. The iron atoms of haemoglobin and haemochromogens react stoicheiometrically with carbon monoxide and the Fe-If -CO link is dissociated by light. When haemgglobin is converted to oxyhaemoglobin formation of the Fe II -0 2 link does not result in oxidation of the iron. However, the iron of haemochromogens (like that of haem) is readily oxidized by oxygen. Such oxidized haemochromogens show only diffuse absorption bands. ~-The current convention is that absorption bands are denoted by roman letters and cytochrome components by italicised letters.
BIOCHEMICAL EDUCATION
October 1973 Vol. 1 No. 4
wield considerable influence in the world of parasitology. He was editor of the leading journal in that field, Parasitology, from 1934 until his death in 1963 and during that period he came to be regarded as perhaps the greatest parasitologist of his time.
Keilin's work on cytochrome revolutionized biochemical thinking. Firstly, he introduced the concept of a respiratory chain without which there could be no understanding of the energy conservation processes that are unique to living cells. Secondly, he stressed the importance of the spatial orientation of chain components in determining the efficiency of hydrogen transport within the cell. Subsequent developments in knowledge of mitochondrial structure and function have underlined the importance of spatial arrangements. Thirdly, he showed that haemoproteins function as intracellular oxidation-reduction catalysts. Hitherto their only known function had been that of carriers of molecular oxygen (e.g. haemoglobin). An immediate consequence of this was Warburg's classical work on the light-sensitive inhibition of respiration of yeast and bacteria by carbon monoxide. He compared the effects of radiation of different wavelengths upon COinhibited yeast and was able (a) to deduce that his respiratory enzyme was a derivative of haematin and Co) to derive the absorption sly, ctrum of its carbon monoxide derivative. Later Keilin and I (5) showed that the a-band of the cytochrome spectrum consists of superimposed bands of two cytochrome components, a and as, and that the latter reacts with cyanide and with carbon monoxide. Furthermore, the absorption spectra of the carbon monoxide derivatives of cytochrome a s and of Warburg's respiratory enzyme were similar enough to allow the conclusion that these two enzymes, and indophenol oxidase, are identical. Since the only physiological function of the enzyme appeared to be the oxidation of cytochrome (6) it was re-named cytochrome oxidase. Subsequently we found that the band c, as observed in cells and tissues, was not at exactly the same wavelength as the a-band of isolated soluble cytochrome c. This led to the discovery of an additional respiratory chain component, cytochrome c l , with absorption bands very close to those of cytochrome c (7). The fact that the respiratory chain could be studied piecemeal stimulated world-wide and rapid advances in the study of intracellular catalysts. Dehydrogenases were purified and characterized; a new group of respiratory catalysts, the fiavoproteins, were characterized and some were found to be components of the respiratory chain; the roles of co-enzymes (now referred to as NAD and NADP) were delineated; and the role of the cytochrome system in oxidative phosphorylation was revealed. At the same time new cytochromes were discovered: some of widespread occurrence; others of limited distr~ution and of unexpected function (e.g. anaerobic metabolism of sulphur bacteria). Although the discovery of cytochrome may be formally dated 1925 it is in fact a continuing process.
To assess the impact of the discovery of cytochrome it is necessary to recall the efforts made during the fifteen years preceding 1925 to understand the mechanism of cell respiration. Two rival schools of thought had developed (l). The dehydrogenase theory, associated with the names of Thunberg and Wieland, proposed that the codtrolling factors were dehydrogenases; enzymes each catalysing the intracellular transfer of pairs of hydrogen atoms from specific metabolites to hydrogen acceptors (e.g. lactate + A "-*pyruvate + AH2). The reduced acceptors wexe assumed to be autoxidizable and to be regenerated by reaction with oxygen. Dehydrogenases had in fact been identified through the ability of tissue extracts to transfer hydrogen atoms from metabolites to an artificial acceptor, such as methylene blue. Though sensitive to urethane such reactions were unaffected by cyanide. Furthermore, the nature of natural acceptors, and whether oxygen itself could act as acceptor, remained uncertain. The alternative view, developed by Warburg, was that intracellular oxidations were controlled by two factors: non-specific forces that led to attachment of metabolites to membrane surfaces within the cell and oxidation of adsorbed metabolites catalysed by a single, universal, respiratory enzyme which was a component of such surfaces. Warburg concluded that the active centre of the respiratory enzyme was iron (which would explain cyanide sensitivity) and that the iron, through alternative oxidation and reduction, catalysed the reaction between oxygen and surface-bound metabolites. This hypothesis ruled out any role, or indeed need, for dehydrogenase~ The two schools appeared to be irreconcilable until Keilin showed that each hypothesis represented part of the truth and that cytochrome was the link between them. Tht~s MacMunn's work is a classical example of a premature discovery. Michael Foster ( 1 8 3 6 1907), the eminent teacher of physiology at Cambridge, wrote: "... there is a time and place for everything, including a new truth. If a discovery is made before its time, it withers up barren, without progeny ...". There was, in present-day jargon, no vacant slot into which MacMunn's work would fit. There is a certain irony in the fact that it was Foster who, a decade earlier, had communicated MacMunn's main paper to the Royal Society. In the years up to 1931 (4) Keilin established the essential structure of the respiratory system as: substrates--* dehydrogenases* -~ cytochrome --* oxidase* * --* 0 2 (arrows representing H-transfer; asterisks indicating points of action of urethane* and cyanide**). Progress became more rapid when yeast was replaced by a suspension of washed heart-muscle particles. This contained the eytochromemediated respiratory system at high concentrations. Its main advantages over yeast were the complete lack of metabolites (enzyme substrates) and the absence of the cell-wall as a diffusion barrier for substrates and inhibitors. With such a preparation Keilin could study the respiratory system in sections, showing for example that dehydrogenases catalyse the transfer of hydrogen atoms initially to eytochrome b. He isolated cytochrome c in soluble form from yeast and determined its properties and its relationship to haemochromogens. He showed that substances which reduced cytochrome c (e.g. cysteine)' underwent oxidation in presence of the heart preparation and oxygen. This oxidation, mediated by cytochromes a and c, was accelerated by adding soluble cytochrome c and it was inhibited by cyanide and by carbon monoxide. Thus oxidation of cysteine proceeded more slowly in a CO/O2 mixture than in the equivalent N2/O2 mixture. However when the cysteine - heartmuscle system in CO/O2 was strongly illuminated the rate of respiration increased to the level observed in N2/O 2. A similar light-reversible inhibition by carbon monoxide had been established for the respiration of bakers' yeast. Keilin also showed that indophenol oxidase, which had been characterized 40 years earlier as an enzyme cataiysing the oxidation by oxygen of a mixture of N, N-dimethyl-p-phenylenediamine and ~-naphthol to a blue indophenol, was identical with the enzyme catalysing the oxidation of cytochrome.
71
In concluding I would fike to return to Gasterophilus intestinalis and the microspectroscope. If we ask: what made possible the discovery of cytochrome; clearly it was the right instrument in the hands of a man of vision. The instrument, a simple microscope accessory, had scarcely changed since MaeMunn's time. The man had the curiosity to venture beyond the organism that had aroused his curiosity into a new field of science. Fortunately for biochemistry the virtues of planned research had yet to be realized. Keilin continued to use this instrument throughout his working life. With its help he could rapidly define a problem and plan the crucial experiments. To answer the problem more sophisticated, spectrophotometric, equipment might have been called in but more often the microspectroscope provided the answers (e.g. ref. 5). The full impact of the discovery of cytochrome rested less upon establishing a widespread existence of this family of pigments (for this much credit must go to MacMunn) than upon the demonstration of its function in living cells. While it is simple to discern this function by Keilin's technique, to d o t h e same with a routine photoelectric spectrophotometer is impossible. This will be abundantly clear to anyone who has attempted to use such an instrument to confirm Keilin's classical experiments on the oxidation and reduction of cytochrome. It is true that spectrophotometers have been devised for studying the kinetics of small and rapid absorption changes in the spectral bands of cytochrome. But such instruments were devised because Keilin's experiments had pointed the way to
(Continued on page 74.]
October 1973 Vol. 1 No. 4
THE DISCOVERY OF CYTOCHROME
E.F. HARTREE A.R.C. Unit of Reproductive Physiology and Biochemistry, 307 Huntingdon Road,
69
i
In these days, when the world of science is under pressure to organize for the pursuit of practical ends, when the scale of scientific endeavour is making the lone furrow an anachronism, it is salutory to recall the single-hande0, achievements of men of science stimulated solely by an urge ~o.understand the living world. The discovery of cytochrome t l ) is a notable example of such achievements not only because of the disarming simplicity of experimental approach by the discoverer, David Kellin, but also because the discovery came at a critical moment. It resolved a serious dilemma that was impeding the evolution of biochemistry from an untidy and rather primitive branch of chemistry into a major scientific discipline: mote succinctly the tranfformation of bio-Chemistry into Biochemistry. Keilin's early life was spent in Warsaw but in 1904, at the age of 17, he began his university career in Li[ge, moving the following year to Paris. One day he happened to take shelter from the rain in a building where a lecture was in progress. This was the beginning of a life-long friendship with the lecturer, Manrice Caullery, and of Keilin's career in biology. At the Sorbonne he read geology, botany, zoology and embryology, while simultaneously carrying out research under Cauilery in parasitology and entomology. By 1915, when he had achieved high reputation as a parasitologist, he came to Cambridge to join George Nuttall (Quick Professor of Biology) and to continue his research on parasitic Diptera. In 1921 Nuttall's group moved t o the newly-built Molteno Institute with which Keilin's name is inseparably linked. At this time Keilin was deeply interested in the striking evolutionary adaptations of dipterous larvae. Many such adaptations are the consequence of a larva's food being situated in a medium deficient in oxygen. Am9ng objects of study were larvae of the fly Gasterophilus intestinalis tl)which parasitize the stomachs of horses, becoming attached to the mucosa by mouth-hooks. The immature larvae are uniformly red but as they grow the pigment becomes localized in trachael ceils which fill the posterior third of the body. These cells, forming integral parts of the larva's respiratory system, each contain an extensive ramification of fine tracheoles which branch off from the trachae. In order to characterize the pigment Keilin used a microspectroscope which is a direct-vision spectroscope ocular designed to replace the eygpiece of a microscope. He placed a living larva above the microscope condenser lens, compressed it lightly with a microscope slide, and illuminated it with a high-intensity light source. The red transmitted light revealed in the microspectroscope the two characteristic absorption bands of oxyhaemoglobin. These bands were however at wavelengths slightly different from those of horse oxyhaemogiobin. The pigment is thus a ~ecific Gasterophllus oxyhaemoglobin. Noting that no tissues of the adult fly are red Keilin was curious about the fate of this oxyhaemogiobin. He therefore examined spectroscopically tissues dissected from the adult. He found that the wing-muscles, which were pale orange-pink by transmitted light, showed four sharp absorption bands. None of these bands corresponded, in terms of wavelength, to the bands of oxyhaemogiobin. He then examined tissues of blow-flies and other insects and without exception he detected in the wing-muscles the same four-banded spectrum. Having cultures of Bacillus subtilis on hand Keilin was inspired to examine them spectroscopically. He scraped the growing cells from agar plates and placed the cheesy mass under the microscope as a thin layer on a glass slide. Again the same four-banded spectrum! This result led him to examine a flattened pellet of baker's yeast - and once more with the same result. It was now clear that the new pigment was not a transformation product of oxyhaemoglobin.
Cambridge CB30JQ, England. Wishing to study the effects of reagents upon the pigment Keilin prepared a thick suspension of yeast by shaking it vigorously with water in a test-tube. Surprisingly, an immediate examination of the suspension revealed only diffuse and scarcely perceptible absorption bands. But after a few seconds' delay the four sharp bands appeared quite suddenly. This remarkable phenomenon could be demonstrated repeatedly with the same sample of yeast suspension: if it was examined immediately after vigorous shaking the bands could not be detected but within 5 - 1 0 seconds, while the spectrum was kept under observation, the four bands reappeared. Keilin's interpretation of these experiments was as follows:- The yeast pigment, to which • he gave the name cytochrome, became oxidised during the shaking in air and in the oxidised form it showed no sharp absorption bands. When shaking was interrupted the respiratory activity of the yeast led to a progressive decrease in the concentration of dissolved oxygen and to reduction of cytochrome, the reduced form of which shows the four-banded spectrum. In other words cytochrome was acting as a respiratory carrier linking oxygen with the oxidizable metabolites in the living yeast cell. Substantial support for this hypothesis soon followed. Thus respiratory poisons (substances that reduce the rate of oxygen consumption by aerated yeast suspensions) also I~locked the spectroscopicaily observed oxidation-reduction cycle of cytochrome. For example, cyanide prevented the oxidation of cytochrome by aeration while urethane greatly delayed the reduction of cytochrome after aeration had ceased. The rate of reduction was also slow in starved yeast, i.e. yeast which, as an aqueous suspension, had been aerated for a long
DAVID KEILIN 1887 - 1963
70
BIOCHEMICAL EDUCATION
period to exhaust stored metabolites. Further study showed that cytochrome was very widely distributed, being present in many animal and plant tissues as well as in yeasts and bacteria. This survey yielded two noteworthy results. Firstly, the cytochrome spectrum showed a remarkably constant pattern: reading from the red end of the spectrum Keilin designated the bands a, b, c, and d. The first three were sharp and prominent while d was fainter and more diffuse as though consisting of several weaker, but clostly situated bands. In bakers' yeast the wavelengths of the band centres were: a, 604 nm; b, 564 nm; c, 550 nm; d, 521 nm. In most other tissues the wavelengths varied only slightly from the yeast values, although wider variations in pattern were found among aerobic bacteria. Several strictly anaerobic bacteria contained no detectable cytochrome. Secondly, there was an obvious relationship between the intensity of absorption bands (i.e. the concentration of cytochrome) in a tissue and the respiratory activity of that tissue. A high cytochrome content was noted in yeast, aerobic bacteria, flight muscles of insects and birds, heart-muscle and brain grey matter of mammals. Detection of cytochrome in mammalian tissue was facilitated if blood was removed by perfusion. Study of two moths provided results of considerabl.e interest. The male winter-moth, Cheimotobia brumata, flies and has well-developed thoracic muscles which are rich in cytochrome. The female" has rudimentary, non-functional wings and the musculature shows a very feeble cytochrome spectrum. However, it was with the wax-moth, Galleria mellonella, that Keflin carried out his most fascinating experiments on the functioning of cytochrome in a living organism. He attached a living specimen to a glass slide with gum arabic, illuminated the thorax from below and studied the transmitted light with the microspectroscope. Periodically the moth would attempt to detach itself by vibrating the wings. While this was happening strong cytochrome bands could be seen. During intervening quiescent periods the absorption bands were very weak. When the immobilized insect was placed in a glass box and a trace of hydrogen cyanide was introduced the insect remained immobile but strong cytochrome bands appeared. Replacing air in the box by nitrogen also caused cytochrome bands to appear. In both cases flushing the box with air led to a return of periodic wing vibration and to the previously observed oxidation and reduction of cytochrome. In discussing his experiments Keilin emphasized that the state of cytochrome in living tissues is a dynamic equilibrium between two reactions, oxidation and reduction, both of which are proceeding rapidly. Thus the reduction of cytochrome in presence of cyanide is not d n a t o any reducing action of cyanide but to an interference with the oxidation process and the establishment of a new equilibrium. Because of its widespread occurrence Keilin scarcely believed that cytochtome, could have remained unnoticed by others. After much searching tl ? he found in old text-books of physiology confusing references to "myohaematin", a pigment which was reputed to occur in muscle and other animal tissues. Following these leads Keilin came a c r ~ papers published in 1884-1887 by C.A. MacMunn ~ " , a medical practitioner and, in his spare time, a spectrolcopist. MacMunn had given the names myohaematin and histohsmatin to pigments characterized by four-banded absorption
*In Ma~lunn's time the term haemochromogen was applied in~inately to reddish breakdown products of haemoglobin that showed prominent absorption bands in the visible spectrum. By 1925 the term was commonly applied to denatmed haemoginbin. Native haemoglobin is a complex of a specific protein, globin, with ferrous protoporphyrin (commonly known as heem). When haemoglobin is denatured, e.g. by heat or alkali, and a r e d U ~ agent is added the remlting complex of h u m and , denatured globin is designated 81obin haemochromogen. Haem freed fro m globin also forms haemochromogens with other denatured proteins. Reducing conditions are essential since haem reacts readily with oxygen to give haematin (i.e. ferric protoporphyrin). The spectrum of haemochromogen consists of two sharp bands of unequal intensity: a strong or-band and, nearer to
October 1973 Vol. 1 No. 4
spectra that he detected in a wide range of animal tismes from sponges to man. He obtained spectra of the oxidized and reduced forms by chemical means (addition of hydrogen peroxide and ammonium sulphide resl~ctively). Although he did not observe rever~ble oxidation and reduction in living tissues he was convinced that the pigments were respiratory carriers. His papers came under attack from some of the "big guns" of physiology who concluded that MacMunn's four-banded spectrum represented no more than a mixture of known haematin pigments (heemoproteins) such as myoglobin and haemoglobin together with haemochromogen derived from these pigments. Such criticilans were accepted by MacMunn's contemporaries and his work was forgotten. Because of his interest in Gasterophilus Keilin had familim~ed himself with the properties, in particular the spectroscopy, of derivatives of haematin: e.g. the oxygenated and deoxygenated forms of haemoglobin arld myoglobin as well as h a e m o d l r o ~ s and related" compounds. Being able to distinguish mixtures of sttch compounds from cytochrome Kenin was not only able to grasp the significance of MacMunn's work but also to unravel the confused and sometimes unwarranted criticisms to which this work had been subjected.* Keilin noticed that when a layer of yeast was left to dry and was examined periodically with the microspectroscope the a-band disappeared first. The b-band d ~ a r e d late~ alid finally the c- and d-bands faded simultaneouS. Extraction of fresh yeast with hot water or dried yeast with cold water yielded soltttions showing band c and a narrowed band d. KeRin concluded that c y t ~ o m e consists of three component pigments, which he designated cytochromes a, b and c, each showing a haemochromogen-type spectrum in the reduced form. t Thus bands a, b, and c are the three strong fl,hands of the three components and band d represents fused weak ~-bands.The components were regarded as "native haemochromollens" since they did not combine with carbon monoxide and were not readily autoxidizable, i.e. they did not become oxidized if shaken with air in presence of a trace of cyanide (the role of cyanide is to inhibit the enzyme cytochrome oxidase - see below). When cytochtome was denatured the absorption bands shifted slightly and the components acquired normal properties of heemochromogens: they became autooxidizable in presenCe of cyanide and they formed spectroscopically recognizable carbon monoxide derivatives. At this stage of his work Keilln published hisJ~i~st paper on cytochrome in the Proceedings of the Royal Society t3.,. The same year saw the beginning of a remarkable period in Keilin's careez. It was in that year that he was appointed university lecturer in parasitology. This involved him in a heavy programme of lectures and practical classes. Within the period 1925-31 he reached the zenith of his career as an experimental parasitologist and published ten papers on the subject. His spare time he devoted to cytochrome, publishing nine papers in this field in the years up to 1931, He was sole author of the first six (3, 4) and R was these that established cytochrome as the keystone of the respiratory mechanism in aerobic cells. Although he subsequently gave up parasitological research he continued to
the blue end of the spectrum, a weak ~-hand. The two bands of oxyhaemogiobin are of nearly equal intensity and are at wavelengths different from those of the derived heemochromogen. The iron atoms of haemoglobin and haemochromogens react stoicheiometrically with carbon monoxide and the Fe-If -CO link is dissociated by light. When haemgglobin is converted to oxyhaemoglobin formation of the Fe II -0 2 link does not result in oxidation of the iron. However, the iron of haemochromogens (like that of haem) is readily oxidized by oxygen. Such oxidized haemochromogens show only diffuse absorption bands. ~-The current convention is that absorption bands are denoted by roman letters and cytochrome components by italicised letters.
BIOCHEMICAL EDUCATION
October 1973 Vol. 1 No. 4
wield considerable influence in the world of parasitology. He was editor of the leading journal in that field, Parasitology, from 1934 until his death in 1963 and during that period he came to be regarded as perhaps the greatest parasitologist of his time.
Keilin's work on cytochrome revolutionized biochemical thinking. Firstly, he introduced the concept of a respiratory chain without which there could be no understanding of the energy conservation processes that are unique to living cells. Secondly, he stressed the importance of the spatial orientation of chain components in determining the efficiency of hydrogen transport within the cell. Subsequent developments in knowledge of mitochondrial structure and function have underlined the importance of spatial arrangements. Thirdly, he showed that haemoproteins function as intracellular oxidation-reduction catalysts. Hitherto their only known function had been that of carriers of molecular oxygen (e.g. haemoglobin). An immediate consequence of this was Warburg's classical work on the light-sensitive inhibition of respiration of yeast and bacteria by carbon monoxide. He compared the effects of radiation of different wavelengths upon COinhibited yeast and was able (a) to deduce that his respiratory enzyme was a derivative of haematin and Co) to derive the absorption sly, ctrum of its carbon monoxide derivative. Later Keilin and I (5) showed that the a-band of the cytochrome spectrum consists of superimposed bands of two cytochrome components, a and as, and that the latter reacts with cyanide and with carbon monoxide. Furthermore, the absorption spectra of the carbon monoxide derivatives of cytochrome a s and of Warburg's respiratory enzyme were similar enough to allow the conclusion that these two enzymes, and indophenol oxidase, are identical. Since the only physiological function of the enzyme appeared to be the oxidation of cytochrome (6) it was re-named cytochrome oxidase. Subsequently we found that the band c, as observed in cells and tissues, was not at exactly the same wavelength as the a-band of isolated soluble cytochrome c. This led to the discovery of an additional respiratory chain component, cytochrome c l , with absorption bands very close to those of cytochrome c (7). The fact that the respiratory chain could be studied piecemeal stimulated world-wide and rapid advances in the study of intracellular catalysts. Dehydrogenases were purified and characterized; a new group of respiratory catalysts, the fiavoproteins, were characterized and some were found to be components of the respiratory chain; the roles of co-enzymes (now referred to as NAD and NADP) were delineated; and the role of the cytochrome system in oxidative phosphorylation was revealed. At the same time new cytochromes were discovered: some of widespread occurrence; others of limited distr~ution and of unexpected function (e.g. anaerobic metabolism of sulphur bacteria). Although the discovery of cytochrome may be formally dated 1925 it is in fact a continuing process.
To assess the impact of the discovery of cytochrome it is necessary to recall the efforts made during the fifteen years preceding 1925 to understand the mechanism of cell respiration. Two rival schools of thought had developed (l). The dehydrogenase theory, associated with the names of Thunberg and Wieland, proposed that the codtrolling factors were dehydrogenases; enzymes each catalysing the intracellular transfer of pairs of hydrogen atoms from specific metabolites to hydrogen acceptors (e.g. lactate + A "-*pyruvate + AH2). The reduced acceptors wexe assumed to be autoxidizable and to be regenerated by reaction with oxygen. Dehydrogenases had in fact been identified through the ability of tissue extracts to transfer hydrogen atoms from metabolites to an artificial acceptor, such as methylene blue. Though sensitive to urethane such reactions were unaffected by cyanide. Furthermore, the nature of natural acceptors, and whether oxygen itself could act as acceptor, remained uncertain. The alternative view, developed by Warburg, was that intracellular oxidations were controlled by two factors: non-specific forces that led to attachment of metabolites to membrane surfaces within the cell and oxidation of adsorbed metabolites catalysed by a single, universal, respiratory enzyme which was a component of such surfaces. Warburg concluded that the active centre of the respiratory enzyme was iron (which would explain cyanide sensitivity) and that the iron, through alternative oxidation and reduction, catalysed the reaction between oxygen and surface-bound metabolites. This hypothesis ruled out any role, or indeed need, for dehydrogenase~ The two schools appeared to be irreconcilable until Keilin showed that each hypothesis represented part of the truth and that cytochrome was the link between them. Tht~s MacMunn's work is a classical example of a premature discovery. Michael Foster ( 1 8 3 6 1907), the eminent teacher of physiology at Cambridge, wrote: "... there is a time and place for everything, including a new truth. If a discovery is made before its time, it withers up barren, without progeny ...". There was, in present-day jargon, no vacant slot into which MacMunn's work would fit. There is a certain irony in the fact that it was Foster who, a decade earlier, had communicated MacMunn's main paper to the Royal Society. In the years up to 1931 (4) Keilin established the essential structure of the respiratory system as: substrates--* dehydrogenases* -~ cytochrome --* oxidase* * --* 0 2 (arrows representing H-transfer; asterisks indicating points of action of urethane* and cyanide**). Progress became more rapid when yeast was replaced by a suspension of washed heart-muscle particles. This contained the eytochromemediated respiratory system at high concentrations. Its main advantages over yeast were the complete lack of metabolites (enzyme substrates) and the absence of the cell-wall as a diffusion barrier for substrates and inhibitors. With such a preparation Keilin could study the respiratory system in sections, showing for example that dehydrogenases catalyse the transfer of hydrogen atoms initially to eytochrome b. He isolated cytochrome c in soluble form from yeast and determined its properties and its relationship to haemochromogens. He showed that substances which reduced cytochrome c (e.g. cysteine)' underwent oxidation in presence of the heart preparation and oxygen. This oxidation, mediated by cytochromes a and c, was accelerated by adding soluble cytochrome c and it was inhibited by cyanide and by carbon monoxide. Thus oxidation of cysteine proceeded more slowly in a CO/O2 mixture than in the equivalent N2/O2 mixture. However when the cysteine - heartmuscle system in CO/O2 was strongly illuminated the rate of respiration increased to the level observed in N2/O 2. A similar light-reversible inhibition by carbon monoxide had been established for the respiration of bakers' yeast. Keilin also showed that indophenol oxidase, which had been characterized 40 years earlier as an enzyme cataiysing the oxidation by oxygen of a mixture of N, N-dimethyl-p-phenylenediamine and ~-naphthol to a blue indophenol, was identical with the enzyme catalysing the oxidation of cytochrome.
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In concluding I would fike to return to Gasterophilus intestinalis and the microspectroscope. If we ask: what made possible the discovery of cytochrome; clearly it was the right instrument in the hands of a man of vision. The instrument, a simple microscope accessory, had scarcely changed since MaeMunn's time. The man had the curiosity to venture beyond the organism that had aroused his curiosity into a new field of science. Fortunately for biochemistry the virtues of planned research had yet to be realized. Keilin continued to use this instrument throughout his working life. With its help he could rapidly define a problem and plan the crucial experiments. To answer the problem more sophisticated, spectrophotometric, equipment might have been called in but more often the microspectroscope provided the answers (e.g. ref. 5). The full impact of the discovery of cytochrome rested less upon establishing a widespread existence of this family of pigments (for this much credit must go to MacMunn) than upon the demonstration of its function in living cells. While it is simple to discern this function by Keilin's technique, to d o t h e same with a routine photoelectric spectrophotometer is impossible. This will be abundantly clear to anyone who has attempted to use such an instrument to confirm Keilin's classical experiments on the oxidation and reduction of cytochrome. It is true that spectrophotometers have been devised for studying the kinetics of small and rapid absorption changes in the spectral bands of cytochrome. But such instruments were devised because Keilin's experiments had pointed the way to
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