376
BIOCHEMICAL SOCIETY TRANSACTIONS
Fay, P. (1965) J. Gen. Microbiol. 39, 11-20 Hoober, J. K., Siekevitz, P. & Palade, G. E. (1969) J. Biol. Chem. 244,2621-2631 Khoja, T. & Whitton, B. A. (1971) Arch. Mikrobiol. 79, 280-282 Kirk, J. T. 0. (1971) Annu. Rev. Biochem. 40, 160-196 Kratz, W.A. & Myers, J. (1955) Amer. J. Bot. 42, 282-287 Lex, M., Dickson, A. E. & Carr, N. G. (1974) Brit. Phycol. J. 9,221 Myers, J. (1940) Plant Physiol. 15, 575-588 Schor, S., Siekevitz, P. & Palade, G. E. (1970) Proc. Nat. Acad. Sci. US.66, 174-180 Senger, H. (1970) PZantu 92,327-332 Senger, H. & Bishop, N. I. (1969) Nature (London) 221,975-979 Simonis, W.& Urbach, W. (1973) Annu. Rev. Plant Physiol. 24, 89-114
The Occurrence of the Pyruvate Dehydrogenase Complex and the Pyruvate-Ferredoxin Oxidoreductase in Blue-Green Bacteria HERMANN BOTHE Ruhr- Universitit Bochum, Lehrstuhl Biochemie der PJlanzen, 0-4630Bochum, Postfach 2148, German Federal Republic Two different reactions are known for the formation of acetyl-CoA and COz from pyruvate and CoA. One is catalysed by the multienzyme pyruvate dehydrogenase complex. The role of the cofactors involved in this system, thiamin pyrophosphate, lipoic acid, FAD and NAD+, is well established. The other oxidative decarboxylation of pyruvate is catalysed by the pyruvateferredoxin oxidoreductase, which has mainly been described in anaerobic bacteria. In this case the remaining electrons are transferred not to NAD+ but to ferredoxin. In blue-green bacteria the nature of the enzyme systems that catalyse the decarboxylation of keto acids with concomitant formation of acyl-coenzymes is uncertain. The pyruvate dehydrogenase complex has not yet been demonstrated [cf. Smith (1973), Tables 1 and 21. Recently a ferredoxin-dependent decarboxylation of pyruvate was demonstrated in Anabaena variabilis (Leach & Carr, 1971) and Anabaena cylindrica (Bothe & Falkenberg, 1973). Extracts from Anabaenacylindricaalsocatalysethe synthesis of pyruvate from COz, acetyl-CoA and reduced ferredoxin, and (with a high rate) the exchange reaction between COz and the carboxyl group of pyruvate. Thus all the reactions typical of the pyruvateferredoxin oxidoreductase have been demonstrated in Anabaena cylindrica. The activity of the pyruvateferredoxin oxidoreductase is about fivefold higher in Anabaena grown on NO3- or atmospheric Nz as compared with cells grown on NH3. From this it was concluded that one physiological function of this enzyme is probably to provide reduced ferredoxin for the reduction of NO3- or Nz to NH3 (Bothe et a/., 1974). The pyruvate dehydrogenase complex, on the other hand, is present in the blue-green bacterium Anacystis nidulans. Extracts from this organism catalyse a pyruvate- and CoA-dependent formation of NADH that is completely blocked by 5mM-arsenite. It was also found in Ankistrodesmus braunii, Chlamydomonas reinhardtii, Micrococcus denitriJcans, Azotobacter vinelandii and Escherichia coli. In contrast, the tests were negative for Anabaena cylindrica, Clostridium pasteurianum, Clostridium kluyveri and Chlorobium thiosulfatophilum, which cleave pyruvate and CoA to acetyl-CoA and COz probably exclusively via the pyruvate-ferredoxin oxidoreductase. The conclusions from the enzymic determinations are supported by the results of a microbiological test on lipoic acid done with a strain of Streptococcus faecalis deficient in this coenzyme (Bothe & Nolteernsting, 1975). High concentrations of lipoic acid were found in Anacystis, Ankistrodesmus, Chlamydomonas, Micrococcus, Azotobacter 1975
377
555th MEETING, ABERYSTWYTH
and E. coli. In contrast, Anabaena cylindrica, Chlorobium, Clostridium pasteurianum and Clostridium kluyveri contain only minute amounts of this coenzyme, and these observat tions also exclude the occurrence of the lipoic acid-dependent pyruvate dehydrogenase complex in the latter organisms. Bothe, H. & Falkenberg, B. (1973) Plant Sci.Lett. 1, 151-156 Bothe, H. & Nolteernsting, U. (1975) Arch. Mikrobiol. 102,53-57 Bothe, H., Falkenberg, B. & Nolteernsting, U. (1974) Arch. Microbiol. 96,291-304 Leach, C . K. & Carr, N. G. (1971) Biochim. Biophys. Acta 245, 165-174 Smith, A. J. (1973) in The Biology of Blue-Green Algae (Carr, N. G . & Whitton, B. A., eds.), pp. 1-38, Blackwell, Oxford I
Two Plant-Type Ferredoxins in Light-Grown and Dark-Grown Cells of the Blue-Green Bacterium Nostoc Strain MAC KENNETH G. HUTSON and LYNDON J. ROGERS Department of Biochemistry and Agricultural Biochemistry, University College of Wales, Aberystwyth, Dyfed S Y23 3D D , U.K.
The iron-sulphur proteins known as ferredoxins play a major role in plant and bacterial photosynthesis (see, e.g., Buchanan & Arnon, 1970). They are small proteins usually with molecular weights of approx. 6000 or 11000, possessing iron and labile sulphide in equimolar amounts of 2 , 4 or 8 per molecule, depending on the source. They exhibit characteristic visible- and u.v.-absorption spectra, have negative redox potentials and a characteristic electron-paramagnetic-resonance signal in the reduced state (Hall et al., 1973). Ferredoxins from blue-green bacteria appear to be of the type characteristic of higher plants; they are of molecular weight 11000, contain two atoms each of iron and labile sulphide, and accept one electron on reduction. Since blue-green bacteria represent an intermediate stage in evolution between the anaerobic photosynthetic bacteria and green plants, study of their ferredoxins might give some insight into the biochemical evolution of photosynthesis. The properties of the ferredoxins from a number of blue-green bacteria have been summarized elsewhere (Andrew et al., 1975), and at the present meeting (Hall et al., 1975). A few blue-green bacteria such as Aphanocapsa 6714, Chlorogloeopsis (Chlorogloea) fritschiiand Nostoc strain MAC are capable both of growth autotrophically in light and heterotrophically in the dark (Fay, 1965; Hoare et al., 1971; Rippka, 1972). The ferredoxins present in cells grown under these different conditions are being isolated in our laboratory and their properties studied. The present communication reports a study of the ferredoxins from Nostoc strain MAC, a filamentous blue-green bacterium of the order Nostocales, originalIy isolated from negatively geotrophic coralloid roots of the cycad Macrozamia lucida L. Johnson (Bowyer & Skerman, 1968). Light-grown or dark-grown Nostoc strain MAC cells (Hoare et al., 1971) in quantities of approx. 140g were harvested and acetone-dried powders (approx. 30g yield) were prepared. Purification of the ferredoxin after extraction of soluble protein was by a sequence involving treatment with deoxyribonuclease and ribonuclease to degrade polynucleotides, removal of protein precipitated by 40 % saturation with (NH&S04, and adsorption on a DEAE-cellulose column and elution by a continuous C1- gradient. An unexpected finding was that both light-grown and dark-grown Nostoc strain MAC produces two types of ferredoxin, designated type 1 and type 11, in approximate ratio 5 : 1, separable by DEAE-cellulose chromatography. These ferredoxins were separately concentrated by DEAE-cellulose chromatography, being obtained in yields of approx. 20mg of type I ferredoxin and 4mg of type I1 ferredoxin. Some of the properties of the VOl. 3 13
BIOCHEMICAL SOCIETY TRANSACTIONS
Fay, P. (1965) J. Gen. Microbiol. 39, 11-20 Hoober, J. K., Siekevitz, P. & Palade, G. E. (1969) J. Biol. Chem. 244,2621-2631 Khoja, T. & Whitton, B. A. (1971) Arch. Mikrobiol. 79, 280-282 Kirk, J. T. 0. (1971) Annu. Rev. Biochem. 40, 160-196 Kratz, W.A. & Myers, J. (1955) Amer. J. Bot. 42, 282-287 Lex, M., Dickson, A. E. & Carr, N. G. (1974) Brit. Phycol. J. 9,221 Myers, J. (1940) Plant Physiol. 15, 575-588 Schor, S., Siekevitz, P. & Palade, G. E. (1970) Proc. Nat. Acad. Sci. US.66, 174-180 Senger, H. (1970) PZantu 92,327-332 Senger, H. & Bishop, N. I. (1969) Nature (London) 221,975-979 Simonis, W.& Urbach, W. (1973) Annu. Rev. Plant Physiol. 24, 89-114
The Occurrence of the Pyruvate Dehydrogenase Complex and the Pyruvate-Ferredoxin Oxidoreductase in Blue-Green Bacteria HERMANN BOTHE Ruhr- Universitit Bochum, Lehrstuhl Biochemie der PJlanzen, 0-4630Bochum, Postfach 2148, German Federal Republic Two different reactions are known for the formation of acetyl-CoA and COz from pyruvate and CoA. One is catalysed by the multienzyme pyruvate dehydrogenase complex. The role of the cofactors involved in this system, thiamin pyrophosphate, lipoic acid, FAD and NAD+, is well established. The other oxidative decarboxylation of pyruvate is catalysed by the pyruvateferredoxin oxidoreductase, which has mainly been described in anaerobic bacteria. In this case the remaining electrons are transferred not to NAD+ but to ferredoxin. In blue-green bacteria the nature of the enzyme systems that catalyse the decarboxylation of keto acids with concomitant formation of acyl-coenzymes is uncertain. The pyruvate dehydrogenase complex has not yet been demonstrated [cf. Smith (1973), Tables 1 and 21. Recently a ferredoxin-dependent decarboxylation of pyruvate was demonstrated in Anabaena variabilis (Leach & Carr, 1971) and Anabaena cylindrica (Bothe & Falkenberg, 1973). Extracts from Anabaenacylindricaalsocatalysethe synthesis of pyruvate from COz, acetyl-CoA and reduced ferredoxin, and (with a high rate) the exchange reaction between COz and the carboxyl group of pyruvate. Thus all the reactions typical of the pyruvateferredoxin oxidoreductase have been demonstrated in Anabaena cylindrica. The activity of the pyruvateferredoxin oxidoreductase is about fivefold higher in Anabaena grown on NO3- or atmospheric Nz as compared with cells grown on NH3. From this it was concluded that one physiological function of this enzyme is probably to provide reduced ferredoxin for the reduction of NO3- or Nz to NH3 (Bothe et a/., 1974). The pyruvate dehydrogenase complex, on the other hand, is present in the blue-green bacterium Anacystis nidulans. Extracts from this organism catalyse a pyruvate- and CoA-dependent formation of NADH that is completely blocked by 5mM-arsenite. It was also found in Ankistrodesmus braunii, Chlamydomonas reinhardtii, Micrococcus denitriJcans, Azotobacter vinelandii and Escherichia coli. In contrast, the tests were negative for Anabaena cylindrica, Clostridium pasteurianum, Clostridium kluyveri and Chlorobium thiosulfatophilum, which cleave pyruvate and CoA to acetyl-CoA and COz probably exclusively via the pyruvate-ferredoxin oxidoreductase. The conclusions from the enzymic determinations are supported by the results of a microbiological test on lipoic acid done with a strain of Streptococcus faecalis deficient in this coenzyme (Bothe & Nolteernsting, 1975). High concentrations of lipoic acid were found in Anacystis, Ankistrodesmus, Chlamydomonas, Micrococcus, Azotobacter 1975
377
555th MEETING, ABERYSTWYTH
and E. coli. In contrast, Anabaena cylindrica, Chlorobium, Clostridium pasteurianum and Clostridium kluyveri contain only minute amounts of this coenzyme, and these observat tions also exclude the occurrence of the lipoic acid-dependent pyruvate dehydrogenase complex in the latter organisms. Bothe, H. & Falkenberg, B. (1973) Plant Sci.Lett. 1, 151-156 Bothe, H. & Nolteernsting, U. (1975) Arch. Mikrobiol. 102,53-57 Bothe, H., Falkenberg, B. & Nolteernsting, U. (1974) Arch. Microbiol. 96,291-304 Leach, C . K. & Carr, N. G. (1971) Biochim. Biophys. Acta 245, 165-174 Smith, A. J. (1973) in The Biology of Blue-Green Algae (Carr, N. G . & Whitton, B. A., eds.), pp. 1-38, Blackwell, Oxford I
Two Plant-Type Ferredoxins in Light-Grown and Dark-Grown Cells of the Blue-Green Bacterium Nostoc Strain MAC KENNETH G. HUTSON and LYNDON J. ROGERS Department of Biochemistry and Agricultural Biochemistry, University College of Wales, Aberystwyth, Dyfed S Y23 3D D , U.K.
The iron-sulphur proteins known as ferredoxins play a major role in plant and bacterial photosynthesis (see, e.g., Buchanan & Arnon, 1970). They are small proteins usually with molecular weights of approx. 6000 or 11000, possessing iron and labile sulphide in equimolar amounts of 2 , 4 or 8 per molecule, depending on the source. They exhibit characteristic visible- and u.v.-absorption spectra, have negative redox potentials and a characteristic electron-paramagnetic-resonance signal in the reduced state (Hall et al., 1973). Ferredoxins from blue-green bacteria appear to be of the type characteristic of higher plants; they are of molecular weight 11000, contain two atoms each of iron and labile sulphide, and accept one electron on reduction. Since blue-green bacteria represent an intermediate stage in evolution between the anaerobic photosynthetic bacteria and green plants, study of their ferredoxins might give some insight into the biochemical evolution of photosynthesis. The properties of the ferredoxins from a number of blue-green bacteria have been summarized elsewhere (Andrew et al., 1975), and at the present meeting (Hall et al., 1975). A few blue-green bacteria such as Aphanocapsa 6714, Chlorogloeopsis (Chlorogloea) fritschiiand Nostoc strain MAC are capable both of growth autotrophically in light and heterotrophically in the dark (Fay, 1965; Hoare et al., 1971; Rippka, 1972). The ferredoxins present in cells grown under these different conditions are being isolated in our laboratory and their properties studied. The present communication reports a study of the ferredoxins from Nostoc strain MAC, a filamentous blue-green bacterium of the order Nostocales, originalIy isolated from negatively geotrophic coralloid roots of the cycad Macrozamia lucida L. Johnson (Bowyer & Skerman, 1968). Light-grown or dark-grown Nostoc strain MAC cells (Hoare et al., 1971) in quantities of approx. 140g were harvested and acetone-dried powders (approx. 30g yield) were prepared. Purification of the ferredoxin after extraction of soluble protein was by a sequence involving treatment with deoxyribonuclease and ribonuclease to degrade polynucleotides, removal of protein precipitated by 40 % saturation with (NH&S04, and adsorption on a DEAE-cellulose column and elution by a continuous C1- gradient. An unexpected finding was that both light-grown and dark-grown Nostoc strain MAC produces two types of ferredoxin, designated type 1 and type 11, in approximate ratio 5 : 1, separable by DEAE-cellulose chromatography. These ferredoxins were separately concentrated by DEAE-cellulose chromatography, being obtained in yields of approx. 20mg of type I ferredoxin and 4mg of type I1 ferredoxin. Some of the properties of the VOl. 3 13
