Photosynthesis Research

, Volume 66, Issue 3, pp 159–175 | Cite as

18O and mass spectrometry in chlorophyll research: Derivation and loss of oxygen atoms at the periphery of the chlorophyll macrocycle during biosynthesis, degradation and adaptation

  • Robert J. Porra
  • Hugo Scheer


Chlorophylls, magnesium-containing tetrapyrrolic pigments of photosynthesis, are widely-distributed in Nature and participate in both light harvesting and in the transduction of light energy to chemical energy for the photosynthetic fixation of carbon dioxide. We briefly discuss the extensive role of various isotopic labelling techniques in elucidating the pathway of tetrapyrrole-pigment biosynthesis and we acknowledge the classic and meticulous research of David Shemin who, approximately 50 years ago, introduced isotopic tracer techniques with 15N and 14C isotopes to study the biosynthesis of the carbon/nitrogen macrocycle of haem, an iron tetrapyrrole. The main focus of this review is the application of mass spectrometry and 18O labelling to the study of the incorporation of oxygen atoms from molecular oxygen or water into the periphery of the chlorophyll macrocycle during biosynthesis and their loss during degradation and light acclimation. In particular, we review the mechanism of formation of the isocyclic ring of chlorophylls, in higher plants, green algae and various photosynthetic bacteria, which concomitantly incurs formation of the 131-oxo group that is present in all photosynthetically-active chlorophylls. In addition we discuss the formation of the ubiquitous 133- and 173-carboxyl groups and also the formation of the 7-formyl group of chlorophyll b and the 3-acetyl group of bacteriochlorophyll a.

adaptation biosynthesis bacteriochlorophyll a formation 3-acetyl group formation chlorophyll b formation degradation 7-formyl group formation isocyclic ring E formation oxygenase- and hydratase-catalysed cyclization origin of 133- and 173-carboxyl groups 


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  1. Avissar YJ and Moberg, PA (1995) The common origins of of the pigments of life early steps of chlorophyll biosynthesis. Photosynth Res 44: 221-242CrossRefGoogle Scholar
  2. Avissar YJ, Ormerod JG and Beale SI (1989) Distribution of δ-aminolevulinic acid biosynthetic pathways among phototropic bacterial groups. Arch Microbiol 151: 513-519PubMedCrossRefGoogle Scholar
  3. Battersby AR, Fookes CJR, McDonald E and Meegan MJ (1978) Biosynthesis of type-III porphyrins: Proof of intact enzymic conversion of the head-to-tail bilane into uro'gen-III by intramolecular rearrangement. J Chem Soc Commun, pp 185-186Google Scholar
  4. Battersby AR and Leeper FJ (1990) Biosynthesis of the pigments of life: Mechanistic studies on the conversion of porphobilinogen to uroporphyrinogen III. Chem Rev 90: 1261-1274CrossRefGoogle Scholar
  5. Bauer CE (1995) Regulation of photosynthesis gene expression. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 1221-1234. Kluwer Academic Publishers, Dordrecht, The NetherlandsGoogle Scholar
  6. Beale SI (1999) Enzymes of chlorophyll biosynthesis. Photosynth Res 60: 43-73CrossRefGoogle Scholar
  7. Beale SI and Castelfranco PA (1973) 14C Incorporation from exogenous compounds into δ-aminolevulinic acid by greening cucumber cotyledons. Biochem Biophys Res Commun 52: 143-149 [see also corrected version published in Vol 53, pp 366–367]PubMedCrossRefGoogle Scholar
  8. Beale SI and Castelfranco PA (1974) The biosynthesis of δ-aminolevulinic acid in higher plants. II. Formation of 14C-δ-aminolevulinic acid from labeled precursors in greening plant tissues. Plant Physiol 53: 297-303.PubMedGoogle Scholar
  9. Beale SI and Weinstein JD (1991) Biosynthesis of 5-aminolevulinic acid in phototropic organisms. In: Scheer H (ed) Chlorophylls, pp 385-406. CRC Press, Boca Raton, FloridaGoogle Scholar
  10. Beale SI, Castelfranco PA and Granick S (1975) The biosynthesis of δ-aminolevulinic acid from the intact carbon skeleton of glutamate in greening barley. Proc Natl Acad Sci USA 72: 2719-2723PubMedCrossRefGoogle Scholar
  11. Bollivar DW and Beale SI (1995) Formation of the isocyclic ring of chlorophylls by isolated Chlamydomonas reinhardtii chloroplasts. Photosynth Res 43: 113-124CrossRefGoogle Scholar
  12. Chereskin BM, Wong YS and Castelfranco PA (1984) In vitro synthesis of the chlorophyll isocyclic ring. Transformation of magnesium-protoporphyrin IX and magnesium-protoporphyrin IX monomethylester into magnesium-2,4-divinylpheoporphyrin a 5. Plant Physiol 70: 987-993Google Scholar
  13. Chunaev AS, Mirnaya ON, Maslov VG and Boschetti A (1991) Chlorophyll b-and loroxanthin-deficient mutants of Chlamydomonas reinhardtii. Photosynthetica 25: 2901-301Google Scholar
  14. Doi M and Shioi Y (1991) Enhancement of denitrifying activity in cells of Roseobacter denitrificans grown acrobically in the light. Plant Cell Physiol 32: 365-370Google Scholar
  15. Ellsworth RK and Aronoff S (1968) Investigations on the biogenesis of chlorophyll a. I. Biosynthesis of Mg-vinylpheoporphine a 5 methylester from Mg-protoporphine IX monomethylester as observed in Chlorella mutants. Arch Biochem Biophys 125: 269-277PubMedCrossRefGoogle Scholar
  16. Ellsworth RK and Aronoff S (1969) Investigations on the biogenesis of chlorophyll a. IV. Isolation and partial characterization of some biosynthetic intermediates between Mg-protoporphine IX monomethyl ester and Mg-vinylpheoporphine a 5 obtained from Chlorella mutants. Arch Biochem Biophys 130: 374-383PubMedCrossRefGoogle Scholar
  17. Folly P and Engel N (1999) Chlorophyll b to chlorophyll a conversion precedes chlorophyll degradation in Hordeum vulgare L. J Biol Chem 274: 21811-21816PubMedCrossRefGoogle Scholar
  18. Fookes CJR and Jeffrey SW (1989) The structure of chlorophyll c 3, a novel marine photosynthetic pigment. J Chem Soc Chem Commun 23: 1827-1828CrossRefGoogle Scholar
  19. Gough, SP, Petersen, BO and Duus, JO (2000) Anaerobic chlorophyll isocyclic ring formation in Rhodobacter capsulatus requires a cobalamine cofactor. Proc Natl Acad Sci USA 97: 6908-6913PubMedCrossRefGoogle Scholar
  20. Hayaishi O (1974) General properties and biological functions of oxygenases. In: Hayaishi O (ed) Molecular Mechanisms of Oxygen Activation, pp 1-28. Academic Press, LondonGoogle Scholar
  21. Holt AS (1959) Reduction of chlorophyllides, chlorophylls and chlorophyll derivatives by sodium borohydride. Plant Physiol 34: 310-314PubMedGoogle Scholar
  22. Hörtensteiner S, Vicentini F and Matile P (1995) Chlorophyll breakdown in senescent cotyledons of rape, Brassica napus L.: Enzymatic cleavage of phaeophorbide a in vitro. New Phytol 129: 237-246CrossRefGoogle Scholar
  23. Ito H and Tanaka A (1996) Determination of activity of chlorophyll b to chlorophyll a conversion during greening of etiolated cucumber cotyledons by using pyrochlorophyllide b. Plant Physiol Biochem 34: 35-40Google Scholar
  24. Iturraspe J, Engel N and Gossauer A (1994) Chlorophyll catabolism: Isolation and structure elucidation of chlorophyll b catabolites in Chlorella protothecoides. Phytochemistry 35: 1387-1390CrossRefGoogle Scholar
  25. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) (1988) Nomenclature of Tetrapyrroles, Recommendations 1986, Eur J Biochem 178: 277-328Google Scholar
  26. Jeffrey, SW (1997) Appendix B: Structural relationships between algal chlorophylls. In: Jeffrey SW, Mantoura RFC and Wright SW (eds) Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods, pp 566-571. UNESCO, ParisGoogle Scholar
  27. Jones OTG (1964) Studies on the structure of a pigment related to chlorophyll a produced by Rhodopseudomonas sphaeroides. Biochem J. 91: 572-576PubMedGoogle Scholar
  28. Jordan PM (1991) The biosynthesis of 5-aminolaevulinic acid and its transformation into uroporphyrinogen III. In: Jordan PM (ed) Biosynthesis of Tetrapyrroles, pp 1-66. Elsevier, AmsterdamGoogle Scholar
  29. Jordan PM (1994) biosynthesis of uroporphyrinogen III: Mechanism of action of porphobilinogen deaminase. In: Chadwick DJ and Ackrill K (eds) The Biosynthesis of Tetrapyrrole Pigments, CIBA Foundation No. 180, pp 70-89. John Wiley and Sons, ChichesterGoogle Scholar
  30. Kannangara CG, Andersen RV, Potoppidan B, Willows R and Von Wettstein D (1994) Enzymic and mechanistic studies on the conversion of glutamate to 5-aminolaevulinate. In: Chadwick DJ and Ackrill K (eds) The Biosynthesis of Tetrapyrrole Pigments, CIBA Foundation No. 180, pp 3-25. John Wiley and Sons, ChichesterGoogle Scholar
  31. Leeper FJ (1994) The evidence for a spirocyclic intermediate in the formation of uroporphyrinogen III by cosynthase. In: Chadwick DJ and Ackrill K (eds) The Biosynthesis of Tetrapyrrole Pigments, CIBA Foundation No. 180, pp 111-123. John Wiley and Sons, ChichesterGoogle Scholar
  32. Mathewson JH and Corwin AH (1961) Biosynthesis of pyrrole pigments: A mechanism for porphobilinogen polymerization. J Am Chem Soc 83: 135-137CrossRefGoogle Scholar
  33. Mayer SM, Gawlita E, Avissar YJ, Anderson VE and Beale SI (1993) Intermolecular nitrogen transfer in the enzymic conversion of glutamate to δ-aminolevulinic acid by extracts of Euglena vulgaris. Plant Physiol 101: 1029-1038PubMedCrossRefGoogle Scholar
  34. Miyashita H, Ikemoto H, Kurano N, Adachi K, Chihara M and Miyachi S (1996) Chlorophyll d as a major pigment. Nature (London) 383: 402CrossRefGoogle Scholar
  35. Nasrulhaq-Boyce A, Griffith WT and Jones OTG (1987) The use of continuous assays to characterize the oxidative cyclase that synthesizes the chlorophyll isocyclic ring. Biochem J 243: 23-29PubMedGoogle Scholar
  36. Oh-hama T, Seto H, Otake N and Miyachi S (1982) 13C-NMR evidence for the pathway of chlorophyll biosynthesis in green algae. Biochem Biophys Res Commun 105: 647-652PubMedCrossRefGoogle Scholar
  37. Oh-hama T, Seto H and Miyachi S (1985a) 13C-nuclear magnetic resonance studies of the biosynthesis of 5-aminolevulinic acid destined for chlorophyll formation in dark-grown Scenedesmus obliquus. Plant Sci 42: 153-158CrossRefGoogle Scholar
  38. Oh-hama T, Seto H and Miyachi S (1985b) 13C-nuclear magnetic resonance studies on bacteriochlorophyll a biosynthesis in Rhodopseudomonas spheroides S. Arch Biochem Biophys 237: 72-79PubMedCrossRefGoogle Scholar
  39. Oh-hama T, Seto H and Miyachi S (1986a) 13C-NMR evidence of bacteriochlorophyll a formation by the C5 pathway in Chromatium. Arch Biochem Biophys 246: 192-198PubMedCrossRefGoogle Scholar
  40. Oh-hama T, Seto H and Miyachi S, (1986b) 13C-NMR evidence of bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris. Eur J Biochem 159: 189-194PubMedCrossRefGoogle Scholar
  41. Okamura K, Takamiya K and Nishimura M (1985) Photosynthetic electron transfer system is inoperative in anaerobic cells of Erythrobacter species strain Och 114. Arch Microbiol 142: 12-17CrossRefGoogle Scholar
  42. Oster U, Tanaka R, Tanaka A and Rüdiger W (2000) Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana. Plant J 21: 306-310CrossRefGoogle Scholar
  43. Porra, RJ (1997) Recent progress in porphyrin and chlorophyll biosynthesis. Photochem Potobiol 65: 492-516Google Scholar
  44. Porra RJ, Klein O and Wright PE (1982) 13C-NMR studies of chlorophyll biosynthesis in higher plants: An unequivocal proof of the participation of the C5 pathway and evidence of a new route for the incorporation of glycine. Biochem Internat 5: 345-350Google Scholar
  45. Porra RJ, Klein O and Wright PE (1983) The proof by 13C-NMR spectroscopy of the predominance of the C5 pathway over the Shemin pathway in chlorophyll biosynthesis in higher plants and of the formation of the methyl ester group of chlorophyll from glycine. Eur J Biochem 130: 509-516PubMedCrossRefGoogle Scholar
  46. Porra RJ, Schäfer W, Cmiel E, Katheder I and Scheer H (1993a) Derivation of the formyl-group oxygen of Chlorophyll b from molecular oxygen in greening leaves of a higher plant (Zea mays). FEBS Lett 323: 31-34PubMedCrossRefGoogle Scholar
  47. Porra RJ, Schäfer W, Cmiel E, Katheder I and Scheer H (1993b) The unexpected reduction of the vinyl group of chlorophyll b by sodium borohydride in methanolic extracts of maize leaves and its inhibition by 8-hydroxyquinoline. Z Naturforsch 48c: 746-748Google Scholar
  48. Porra RJ, Schäfer W, Cmiel E, Katheder I and Scheer H (1994) The derivation of the formyl group oxygen of chlorophyll b in higher plants from molecular oxygen: Achievement of high enrichment of the 7-formyl group oxygen from 18O2 in greening maize leaves. Eur J Biochem 219: 671-679PubMedCrossRefGoogle Scholar
  49. Porra RJ, Schäfer W, Katheder I and Scheer H (1995) The derivation of the oxygen atoms of the 131-oxo and 3-acetyl groups of bacteriochlorophyll a from water in Rhodobacter sphaeroides cells adapting from respiratory to photosynthetic conditions: Evidence for an anaerobic pathway for the formation of isocyclic ring E. FEBS Lett 371: 21-24PubMedCrossRefGoogle Scholar
  50. Porra RJ, Schäfer W, Gad'on N, Katheder I, Drews G and Scheer H (1996) Origin of the two carbonyl oxygens of bacteriochlorophyll a: demonstration of two different pathways for the formation of ring E in Rhodobacter sphaeroides and Roseobacter denitrificans, and a common hydratase mechanism for 3-acetyl group formation. Eur J Biochem 239: 85-92PubMedCrossRefGoogle Scholar
  51. Porra RJ, Pfündel E and Engel N (1997) Metabolism and function of photosynthetic pigments. In: Jeffrey SW, Mantoura RFC and Wright SW (eds) Phytoplankton pigments in Oceanography: Guidelines and Methods, pp 85-126. UNESCO, ParisGoogle Scholar
  52. Porra RJ, Urzinger M, Winkler J, Bubenzer C and Scheer H (1998) Biosynthesis of the 3-acetyl and 131-oxo groups of bacteriochlorophyll a in the facultative aerobic bacterium, Rhodovulum sulfidophilum: The presence of both oxygenase and hydratase pathways for isocyclic ring formation. Eur J Biochem 257: 185-191PubMedCrossRefGoogle Scholar
  53. Scheer H (1991) Chemistry of chlorophylls. In: Scheer H (ed) Chlorophylls, pp 3-30. CRC Press, Boca Raton, FloridaGoogle Scholar
  54. Scheumann V, Schoch S, Helfrich M and Rüdiger W (1996a) Reduction of the formyl group of Zn-pheophytin b in vitro and in vivo: A model for the chlorophyll b to a transformation. Z Naturforsch 51c: 185-194Google Scholar
  55. Scheumann V, Ito H, Tanaka A, Schoch S and Rüdiger W (1996b) Substrate specificity of chlorophyll(ide) b reductase in etioplasts of barley (Hordeum vulgare L. Eur J Biochem 242: 163-170PubMedCrossRefGoogle Scholar
  56. Schmid R and Shemin D (1955) The enzymatic formation of porphobilinogen from δ-aminolevulinic acid and its conversion to protoporphyrin. J Am Chem Soc 77: 506-508CrossRefGoogle Scholar
  57. Schneegurt MA and Beale SI (1992) Origin of the chlorophyll b formyl oxygen in Chlorella vulgaris. Biochemistry 31: 11677-11683PubMedCrossRefGoogle Scholar
  58. Shemin D (1956) The biosynthesis of porphyrins. In: Graff S (ed) Essays in Biochemistry, pp 241-248. Wiley, New YorkGoogle Scholar
  59. Shemin D and Rittenberg D (1946a) The biological utilization of glycine for the synthesis of protoporphyrin of hemoglobin. J Biol Chem 166: 621-625Google Scholar
  60. Shemin D and Rittenberg D (1946b) The life span of the human red blood cell. J Biol Chem 166: 627-636Google Scholar
  61. Shioi Y, Doi M, Arata H and Takamiya K (1988) A denitrifying activity in an aerobic photosynthetic bacterium, Erythrobacter sp. Strain Och 114. Plant Cell Physiol 29: 861-865Google Scholar
  62. Shlyk AA (1971) Biosynthesis of chlorophyll b. Ann Rev Plant Physiol 22: 169-184CrossRefGoogle Scholar
  63. Simpson DJ and Smith KM (1988) Structures and transformations of the bacteriochlorophylls e and their bacteriopheophorbides. J Am Chem Soc 110: 1753-1758CrossRefGoogle Scholar
  64. Smith KM and Simpson DJ (1986) Stereochemistry of the bacteriochlorophyll-e homologues. J Chem Soc Chem Commun 1682-1684Google Scholar
  65. Stark WM, Baker MG, Raithby PR, Leeper FJ and Battersby AR (1985) The spiro intermediate proposed for biosynthesis of the natural porphyrins: Synthesis and properties of its macrocycle. J Chem Soc Commun: 1294-1296Google Scholar
  66. Stark WM, Hart GJ and Battersby AR (1986) Synthetic studies on the proposed spiro intermediate for biosynthesis of the natural porphyrins: Inhibition of cosynthase. J Chem Soc Commun, pp 465-467Google Scholar
  67. Tanaka A, Ito H, Tanaka R, Tanaka NK and Okada K (1998) Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a. Proc Natl Acad Sci USA 95: 12719-12723PubMedCrossRefGoogle Scholar
  68. Walker CJ, Mansfield KE, Smith KM and Castelfranco PA (1989) Incorporation of atmospheric oxygen into the carbonyl functionality of the protochlorophyllide isocyclic ring. Biochem J 257: 599-60PubMedGoogle Scholar
  69. Wittenberg J and Shemin D (1950) The location in protoporphyrin of the carbon atoms derived from the α-carbon of glycine. J Biol Chem 185: 103-116PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  1. 1.Botanisches Institutder Ludwig-Maximilians UniversitätMünchenGermany
  2. 2.Division of Plant IndustryCSIROCanberraAustralia
  3. 3.Botanisches Institutder Ludwig-Maximilians UniversitätMünchenGermany

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