Department of Pharmacology and Toxicology, College of Pharmacy, and Environmental and Occupational Health Science Institute, Rutgers University, 681 Frelinghuysen Road, Piscataway, New Jersey 08854
The concepts that underlie the role of oxygen in respiration have evolved over the last two centuries (Keilin, 1966). Antoine Lavoisier (17431794) argued that respiration is the slow burning of carbon derived from the blood, which occurs in the lungs. During the process, oxygen is taken up and carbonic acid produced. Claude Bernard (18131878) suggested that respiration occurs by a process that he called "respiratory fermentation," i.e., enzymatically, within the tissues of the body. The idea that heme proteins played a role in respiration originated with the studies of C. S. McMunn (18521911), who examined specimens of tissue from almost every phylum, ranging from coelenterates to man, using a microspectroscope (McMunn, 1884
). He took care to remove blood from his samples in those species where it interfered with his observations. In all tissues he found a pigment, which he termed myohaematin (in muscles) or histohaematin (in other tissues), and which displayed a common absorption spectrum. He demonstrated that the bands were "...intensified by reducing agents and enfeebled by oxidizing agents; they accordingly appear to be capable of oxidation and reduction and are, therefore, respiratory ...hence, the histohaematins are concerned in the internal respiration of the tissues and organs of invertebrate and vertebrate animals." Despite the fact that this was perhaps the most important statement in the eventual development of our understanding of respiration, his work was criticized by the great German biochemist Hoppe-Seyler, who claimed that histohaematin was a breakdown product of hemoglobin. McMunn never recovered from this attack and the subject lay dormant for 25 years.
David Keilin (18871963) was a parasitologist at Cambridge University who was interested in the anatomy of the respiratory system in dipterous flies. While studying the abdominal muscle of an adult fly under the microspectroscope, he rediscovered the four-banded pigment. He studied many other species and, given the developments in biology over the intervening 40 years, was able to deflate the claims of Hoppe-Seyler and vindicate the work of McMunn. He demonstrated that the four bands represented the visible light absorption spectra of three pigments that he called cytochromes. Although cytochromes a, b, and c appeared to play a role in the respiratory process, they did not appear to be the site at which oxygen reacts. The oxygen-activating enzyme had to be auto-oxidizable and capable of reacting with carbon monoxide when reduced, and cyanide when oxidized. Eventually, Keilin called the oxygen-activating enzyme cytochrome oxidase.
The direct demonstration of the reaction of oxygen with cytochrome oxidase arose out of a report by Haldane and Smith (1896), who showed that carbon monoxide could displace oxygen from hemoglobin, but when light was shone on the HbCO complex, it dissociated. Otto Warburg (18831970) of the Kaiser Wilhelm Institut fur Zellphysiologie in Berlin, using the respirometer that bears his name, demonstrated that oxygen consumption in yeast was inhibited by carbon monoxide in the dark, but when light was shown on the incubation vessel, the inhibition was reversed photochemically (Warburg, 1932). He went on to demonstrate that when performing the experiment at different wavelengths of light from the ultraviolet through the visible spectrum, the degree to which the inhibition was reversed depended upon the wavelength of the light. Furthermore, a comparison of the reversibility of the inhibition plotted against the wavelength yielded a figure, called the photochemical action spectrum, that replicated the absorption spectrum of the cytochrome oxidase-carbon monoxide complex. The combined results of the studies of Keilin and Warburg, and the many related results from the laboratories of their colleagues, have led to our current understanding of mitochondrial electron transport as the basis for cellular respiration.
At the time that Warburg was studying the photochemical properties of cytochrome oxidase, there was a young biochemist at the Kaiser Wilhelm Institut named Otto Rosenthal who became thoroughly familiar with the photochemical action spectrum technique. When he was forced to leave Germany in the 1930s, Dr. Rosenthal came to the Harrison Department of Surgical Research at the University of Pennsylvania. In a collaboration with David Y. Cooper aimed at determining the mechanism by which the adrenals synthesized steroids, they focused on the C-21 hydroxylation of 17-hydroxyprogesterone in adrenal cortical microsomes. S. Narasimhulu showed that the addition of a detergent, Triton X-100, clarified the otherwise optically dense microsomal suspensions and facilitated the spectrophotometric study of the microsomal heme proteins. The addition of the substrate resulted in a change in the spectrum of the heme proteins that has been termed a Type I reaction. Cytochrome b5 was a known microsomal heme protein and was suspected of playing a role in steroid hydroxylation. A discussion with Ronald W. Estabrook led to his participation in the project and the use of an instrument developed at the Johnson Foundation called the Yang-Chance spectrophotometer, in which it what we now recognize as the signature of the cytochrome P450-CO complex was soon observed. The spectrum resembled that observed by Garfinkel (1958) and Klingenberg (1958) in pig and rat liver microsomes, respectively, and which was subsequently used by Omura and Sato (1964) to help identify cytochrome P450 as a heme protein.
To demonstrate that the CO-binding pigment was, in fact, the enzyme involved in C-21 hydroxylation, they took advantage of Rosenthal's familiarity with the photochemical action spectrum technique. C-21 hydroxylation was inhibited by CO; however, the extent of inhibition was determined not by the absolute concentration of CO, but by the ratio of CO/O2, as it is a competitive inhibition. When the incubation vessels were exposed to light at an array of wavelengths, the inhibition could be reversed at those wavelengths that correspond to the range in which the CO-bound heme protein absorbed light. Thus, it was demonstrated that cytochrome P450 was the oxidase that performed the C-21 hydroxylation. It is significant that this report was published in a festschrift honoring the 80th birthday of Otto Warburg (Estabrook et al., 1963).
Rosenthal, Cooper, and Estabrook (Cooper et al., 1965) went on to study the metabolism of codeine, monomethyl-4-aminopyrine, and acetanilide, and found them to be inhibited by carbon monoxide; the CO inhibition was reversed by yielding the same action spectrum, demonstrating that cytochrome P450 is the oxygen-activating enzyme in xenobiotic metabolism as well as in steroid hydroxylation.
The application of the photochemical action spectrum technique to identify the oxygen-activating enzyme in xenobiotic metabolism was possible because of the familiarity with the approach by Rosenthal and the enthusiastic search for the C-21 hydroxylase by Cooper, Estabrook, and Rosenthal. An apparatus in which these studies could be done had to be constructed; once it was available, a number of investigators came to the laboratory in Philadelphia to determine whether the substrates they were studying were also metabolized by cytochrome P450. The results of these studies broadened our appreciation that the biological transport, storage, and utilization of oxygen is, in most cases, mediated by heme proteins.
NOTES
1 To whom correspondence should be addressed. Fax: (732) 445-0119. E-mail: rsnyder{at}eohsi.rutgers.edu.
REFERENCES
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