Cytochrome c Organism Cultural conditions content 0*moles X
10~4/mg. protein)
Pseudomonas aeruginosa High-speed shaking 0.74
Pseudomonas aeruginosa Low-speed shaking 1.03
Pseudomonas aeruginosa Anaerobic 2.90
Micrococcus denitrificans High-speed shaking 0.37 Micrococcus denitrificans Low-speed shaking 0.99
Micrococcus denitrificans Anaerobic 2.14
Pseudomonas fiuorescensf non- High-speed shaking 0.19
denitrifying strain Low-speed shaking 0.37
Additional evidence for a functional role of cytochromes in nitrate re-duction comes from physiological studies. T h e cytochrome c content of a number of nitrate-reducing organisms has been studied as a function of varying oxygen tension, including complete anaerobiosis with n i t r a t e as oxidant.7 1 D a t a typical of the results obtained are shown in T a b l e V I . I t is seen t h a t by far the largest cytochrome c content is obtained from cells grown under conditions which permit the nitrate-reducing system to func-tion optimally.
I t has been shown in earlier work, using P. fluoresceins?* t h a t the cyto-chrome content of pseudomonads decreases in response to strong aeration.
Similar results are not obtained with certain yeasts, however, which can be shown to synthesize large amounts of cytochrome in response to a e r a t i o n .8 6 There have been a number of investigations demonstrating great v a r i a -tion in the cytochrome content of bacteria grown under different cultural conditions. N o general correlation can be made between degree of anaero-biosis and cytochrome content. T h e fact t h a t Azotobacter chroococcum can be shown to form large amounts of cytochrome, detected spectroscopically, merely by allowing washed resting cells to stand overnight in the cold (see review by L. Smith, 1954), is suggestive t h a t the heme proteins can be synthesized from endogenous reserves, or alternatively t h a t p a t h w a y s exist for enzymic interconversion of heme proteins and their prosthetic groups. This latter process might provide a useful regulatory mechanism for a crossover between various types of anaerobic and aerobic electron transport. I n this connection, it would be desirable to conduct studies on variation in content or composition of various heme proteins as a function of change from d a r k aerobic to light anaerobic metabolism in R. rubrum.
C. SULFATE REDUCTION
A number of studies1 0* 8 7« 8 8 have implicated a cytochrome c in sulfate reduction by D. desulfuricans. If precautions are taken to remove the H2S formed on reduction of the substrates, it is possible to show t h a t the purified cytochrome can act as a carrier in the reduction of thiosulfate, t e t r a -thionate, and dithionite by molecular hydrogen and either detergent-treated cells or cell-free extracts of D. desulfuricans. I n addition, these preparations are capable of carrying out a rapid "knallgas" (oxy-hydro-gen) reaction mediated by the hydrogenase and cytochrome c3. Since D.
desulfuricans is an obligate anaerobe, it is of interest t h a t the rate of this reaction is more rapid t h a n the rate of sulfate reduction. This suggests a mechanism for growth inhibition in these organisms analogous to t h a t which is implicated by results with photoautotrophic bacteria, namely, t h a t a nonphosphorylative oxygen reaction can interfere with the normally used electron transport system.
T h e low potential dye, benzyl viologen, can replace the cytochrome in the system coupling hydrogenase to reduction of the various sulfur com-pounds. Hence some caution must be observed in assigning a specific role for the cytochrome in the sulfate-reducing system.
Recently, H . D . Peck, J r .9 0 has obtained a cell-free system from hetero-trophically-grown Desulfovibrio, which can reduce both sulfates and sul-fites, using molecular hydrogen as reductant. A T P is required and evidence is presented implicating adenosine-5'-phosphosulfate as substrate for the sulfate reduction. As with other systems from the anaerobes, the electron transport system from D. desulfuricans appears to be both cyanide- and carbon monoxide-insensitive. The cytochrome c in this organism accounts for 0.3% of the weight of dried bacterial cells.
When D. desulfuricans is grown in iron-deficient p y r u v a t e media, it contains no detectable cytochrome c3 and in addition is unable to reduce sulfate. T h e content of hydrogenase and of the green pigment, desulfo-viridin, is reduced five- and twofold, respectively. I n sharp contrast to D.
desulfuricans, no cytochromes are found in D. thermodesulfuricans, the thermophilic sulfate reducer. Furthermore, when D. thermodesulfuricans strains are selected for growth at 30° C , they still contain no cytochrome.
Analogous results are obtained with strains of D. desulfuricans selected for growth at 50° C.; under these circumstances the original cytochrome complement of the cells is retained. F r o m these results, then, it appears t h a t although cytochrome deficiency in D. desulfuricans is associated with the inability to reduce sulfate, this is not true for the thermophilic strains.
T h e finding t h a t certain strains of D. desulfuricans can grow in sulfate-deficient pyruvate media indicates t h a t the sulfate p a t h w a y for electron transport is not an obligatory one in these organisms, in contrast to the
photosynthetic autotrophs, which so far have never been grown fermenta-tively in t h e dark.*
T h e presence of large amounts of nonheme iron in t h e photophosphoryl-ating particles from t h e photosynthetic autotroph Chromatium h a s been mentioned (see Section I I , B ) . I t is noteworthy t h a t cells of D. desulfuri-cans have also been found to contain appreciable amounts of nonheme iron. When t h e u p t a k e of iron b y certain strains was studied it was found t h a t although over 9 0 % of t h e added iron w a s assimilated, less t h a n 1%
of it could be accounted for as cytochrome, a n d no other hematin compounds could be detected in t h e cells. This additional iron cannot be a s -cribed t o t h e presence of metalloflavins, because t h e flavin content of t h e Chromatium p a r t i c l e s1 6 or of D. desulfuricans89 is not high enough t o bind t h e very large amounts of iron in t h e organisms. I t seems likely t h a t a search for additional iron-binding compounds in these anaerobes would be rewarding. Some preliminary s t u d i e s6 3 in which growth media for R.
rubrum were supplemented with F e5 9- l a b e l e d iron have shown the presence of large amounts of nondialyzable, nonheme iron in soluble protein frac-tions of t h e cells.
V. Conclusions
So far, there is not enough evidence t o justify t h e conclusion t h a t t h e electron transport systems of m a n y anaerobic bacteria differ in a n y major w a y from those of aerobes, either structurally or functionally. A large number of heme proteins have now been isolated from various anaerobic bacteria, and although none of these compounds h a s been shown capable of reacting directly with its physiological oxidant, t h e evidence in favor of functional roles for heme protein mediators of electron transport in these organisms is very convincing.
I t is worth considering w h a t evidence exists for heme protein catalysis of oxygen reactions of m a m m a l i a n cells. Generally, "cytochrome oxidase"
is regarded as an enzyme capable of reacting with molecular oxygen; how-ever, this reaction has y e t t o be demonstrated unequivocally. T h e enzyme of aerobic tissue capable of activating molecular oxygen remains t o be identified conclusively as a heme protein, just as do those enzymes which activate nitrate, sulfate, and other anaerobic oxidants.
T h e photoautotrophic bacteria are extreme obligate anaerobes and will not grow under relatively anaerobic conditions normally used t o grow other anaerobic bacteria, such as t h e Clostridia, for example. This leads to t h e interesting suggestion t h a t t h e presence of a cytochrome system in
* The anomalous status of the thermophilic sulfate-reducers has been clarified by confirmation (J. Postgate, private communication) of findings by Campbell et al.89* that the so-called thermophilic strains of Desulfovibno are in reality clostridial species.
the former organisms is responsible for their extreme anaerobic character.
I t is now known t h a t the cytochromes of these bacteria are low potential, extremely autoxidizable heme proteins and t h a t their phosphorylation re-actions are inhibited by oxygen. T h e fermentative Clostridia m a y be able to tolerate lesser degrees of anaerobiosis because their electron transport systems, lacking cytochromes, are relatively oxygen-insensitive.
I t has been tacitly assumed throughout this chapter t h a t some sort of an electron transport " c h a i n " m a y exist in the organisms discussed, through which electron flow occurs in a graded series of reactions leading to reduc-tion of the electron acceptor. However, it should be equally clear t h a t the existence of an array of spectroscopically differentiable cellular heme pro-teins analogous to t h a t detected in aerobic mammalian cells has not been demonstrated. Instead there are found large concentrations of cytochrome spectroscopically resembling cytochrome c. When some of these compounds are isolated and subjected to extensive purification and analyses, variant cytochrome c types can be found in the preparations which differ in m a n y physicochemical properties, and can be sometimes physically separated into different closely related proteins. I t seems a distinct possibility, there-fore, t h a t electron flow in these systems m a y t a k e place through a series of heme proteins which are electrochemically different, but spectroscopi-cally identical, or very similar. If this suggestion is true, the need to search for heme proteins having electrochemical potentials higher or lower t h a n t h a t found in the cytochromes c would be eliminated. T h e isolated m a t e -rials would represent an "average potential" of a large number of electro-chemically different heme proteins, all of which are spectroscopically in-distinguishable. Evidence supporting this suggestion is provided by the studies cited previously, in which it has been possible to identify a number of different reactive species in the cytochrome c in Chromatium cells by observing polyphasic effects in the rates of reduction and oxidation of the c cytochrome complex found in this organism.
I t should be apparent from the studies recorded in this chapter t h a t m a n y challenging problems await investigation by microbiologists and chemists in the area of electron transport phenomena characteristic of anaerobiosis. I t is to be hoped t h a t a great expansion in research in this area of microbiology occurs in the years immediately ahead. I t is pos-sible t h a t such activity will yield results which will in addition shed new light on m a n y problems of m a m m a l i a n respiration. A t the least, a better appreciation of the m a n y functional consequences inherent in the struc-tural union of heme and proteins should be attained.
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Heme proteins:
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