Sulfate radicals enable a non-enzymatic Krebs cycle precursor


The evolutionary origins of the Krebs cycle (tricarboxylic acid cycle) are not currently clear. Despite the existence of a simple non-enzymatic Krebs cycle catalyst being dismissed only a few years ago as ‘an appeal to magic’, citrate and other intermediates have since been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify a metabolism-like non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate compounds in a reaction mixture that orients on the typical components of Archaean sediment. Krebs cycle intermediates were found to be stable in water and in the presence of most molecule species, including simple iron sulfate minerals. However, in the presence of sulfate radicals generated from peroxydisulfate, the intermediates underwent 24 interconversion reactions. These non-enzymatic reactions covered the critical topology of the oxidative Krebs cycle, the glyoxylate shunt and the succinic-semialdehyde pathway. Assembled in a chemical network, the reactions achieved over 90% carbon recovery. Our results show that a non-enzymatic precursor of the Krebs cycle is biologically sensible, efficient, and forms spontaneously in the presence of sulfate radicals.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: TCA intermediates were stable in water but showed reactivity in the presence of transition metals frequently found in Archaean sediments.
Figure 2: Peroxydisulfate enables the non-enzymatic interconversion of TCA intermediates.
Figure 3: Non-enzymatic Krebs-cycle-like reactions in the presence of peroxydisulfate and peroxydisulfate/ferrous sulfide.
Figure 4: Peroxydisulfate enables TCA-like reactivity by providing sulfate radicals.


  1. 1

    Krebs, H. A. & Johnson, W. A. The role of citric acid in intermediate metabolism in animal tissues. FEBS Lett. 117(suppl.), K1–K10 (1980).

    Article  PubMed  Google Scholar 

  2. 2

    Huynen, M. A., Dandekar, T. & Bork, P. Variation and evolution of the citric-acid cycle: a genomic perspective. Trends Microbiol. 7, 281–291 (1999).

    CAS  Article  PubMed  Google Scholar 

  3. 3

    Meléndez-Hevia, E., Waddell, T. G. & Cascante, M. The puzzle of the Krebs citric acid cycle: assembling the pieces of chemically feasible reactions, and opportunism in the design of metabolic pathways during evolution. J. Mol. Evol. 43, 293–303 (1996).

    Article  PubMed  Google Scholar 

  4. 4

    Wachtershauser, G. Evolution of the first metabolic cycles. Proc. Natl Acad. Sci. USA 87, 200–204 (1990).

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Orgel, L. E. The implausibility of metabolic cycles on the prebiotic Earth. PLoS Biol. 6, e18 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Victor, S., Barry, H., Alexandra, W., Eloi, C. & Nick, L. The origin of life in alkaline hydrothermal vents. Astrobiology 16, 181–197 (2016).

    Article  Google Scholar 

  7. 7

    Hud, N. V., Cafferty, B. J., Ramanarayanan, K. & Williams, L. D. The origin of RNA and ‘My Grandfather’s Axe’. Chem. Biol. 20, 466–474 (2013).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Brasen, C., Esser, D., Rauch, B. & Siebers, B. Carbohydrate metabolism in Archaea: current insights into unusual enzymes and pathways and their regulation. Microbiol. Mol. Biol. Rev. 78, 89–175 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  9. 9

    Morowitz, H. J., Kostelnik, J. D., Yang, J. & Cody, G. D. The origin of intermediary metabolism. Proc. Natl Acad. Sci. USA 97, 7704–7708 (2000).

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Smith, E. & Morowitz, H. J. Universality in intermediary metabolism. Proc. Natl Acad. Sci. USA 101, 13168–13173 (2004).

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Brilli, M. & Fani, R. in Cellular Origin and Life in Extreme Habitats and Astrobiology Vol. 7 (eds Seckbach, J., Chela-Flores, J., Owen, T. & Raulin, F.) 129–132 (Springer, 2004).

    Google Scholar 

  12. 12

    Shapiro, R. A replicator was not involved in the origin of life. IUBMB Life 49, 173–176 (2000).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Zubarev, D. Y., Rappoport, D. & Aspuru-Guzik, A. Uncertainty of prebiotic scenarios: the case of the non-enzymatic reverse tricarboxylic acid cycle. Sci. Rep. 5, 8009 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Cooper, G., Reed, C., Nguyen, D., Carter, M. & Wang, Y. Detection and formation scenario of citric acid, pyruvic acid, and other possible metabolism precursors in carbonaceous meteorites. Proc. Natl Acad. Sci. USA. 108, 14015–14020 (2011).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Zhang, X. V. & Martin, S. T. Driving parts of Krebs cycle in reverse through mineral photochemistry. J. Am. Chem. Soc. 128, 16032–16033 (2006).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    Keller, M. A., Turchyn, A. V. & Ralser, M. Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in a plausible Archaean ocean. Mol. Syst. Biol. 10, 725 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Keller, M. A. et al. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway. Sci. Adv. 2, e1501235 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Rouxel, O. J., Bekker, A. & Edwards, K. J. Iron isotope constraints on the Archaean and Paleoproterozoic ocean redox state. Science 307, 1088–1091 (2005).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Saito, M. A., Sigman, D. M. & Morel, F. M. M. The bioinorganic chemistry of the ancient ocean: the co-evolution of cyanobacterial metal requirements and biogeochemical cycles at the Archaean–Proterozoic boundary? Inorganica Chim. Acta 356, 308–318 (2003).

    CAS  Article  Google Scholar 

  20. 20

    Canfield, D. E., Habicht, K. S. & Thamdrup, B. The Archaean sulfur cycle and the early history of atmospheric oxygen. Science 288, 658–661 (2000).

    CAS  Article  PubMed  Google Scholar 

  21. 21

    Belmonte, L. & Mansy, S. S. Metal catalysts and the origin of life. Elements 12, 413–418 (2016).

    CAS  Article  Google Scholar 

  22. 22

    Zerkle, A. L. Biogeochemical signatures through time as inferred from whole microbial genomes. Am. J. Sci. 305, 467–502 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Siegel, B. & Lanphear, J. Iron-catalyzed oxidative decarboxylation of benzoylformic acid. J. Am. Chem. Soc. 101, 2221–2222 (1979).

    CAS  Article  Google Scholar 

  24. 24

    Tong, W.-H. & Rouault, T. A. Metabolic regulation of citrate and iron by aconitases: role of iron-sulfur cluster biogenesis. Biometals 20, 549–564 (2007).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Kolthoff, I. M. & Miller, I. K. The chemistry of persulfate. I. The kinetics and mechanism of the decomposition of the persulfate ion in aqueous medium 1. J. Am. Chem. Soc. 73, 3055–3059 (1951).

    CAS  Article  Google Scholar 

  26. 26

    Sra, K. S., Thomson, N. R. & Barker, J. F. Persistence of persulfate in uncontaminated aquifer materials. Environ. Sci. Technol. 44, 3098–3104 (2010).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Peyton, G. R. The free-radical chemistry of persulfate-based total organic carbon analyzers. Mar. Chem. 41, 91–103 (1993).

    CAS  Article  Google Scholar 

  28. 28

    Liang, C., Chenju, L., Yi-Yu, G., Yi-Chi, C. & Yi-Jhen, W. Oxidative degradation of MTBE by pyrite-activated persulfate: proposed reaction pathways. Ind. Eng. Chem. Res. 49, 8858–8864 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Liang, C., Bruell, C. J., Marley, M. C. & Sperry, K. L. Persulfate oxidation for in situ remediation of TCE. I. Activated by ferrous ion with and without a persulfate–thiosulfate redox couple. Chemosphere 55, 1213–1223 (2004).

    CAS  Article  PubMed  Google Scholar 

  30. 30

    Teel, A. L., Ahmad, M. & Watts, R. J. Persulfate activation by naturally occurring trace minerals. J. Hazard. Mater. 196, 153–159 (2011).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Herrman, H. On the photolysis of simple anions and neutral molecules as sources of O/OH, SOx and Cl in aqueous solution. Phys. Chem. Chem. Phys. 9, 3935–3964 (2007).

    Article  Google Scholar 

  32. 32

    Liang, C., Chenju, L. & Bruell, C. J. Thermally activated persulfate oxidation of trichloroethylene: experimental investigation of reaction orders. Ind. Eng. Chem. Res. 47, 2912–2918 (2008).

    CAS  Article  Google Scholar 

  33. 33

    Ahmad, M. et al. Oxidative and reductive pathways in iron-ethylenediaminetetraacetic acid–activated persulfate systems. J. Environ. Eng. 138, 411–418 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Schönheit, P., Wolfgang, B. & Martin, W. F. On the origin of heterotrophy. Trends Microbiol. 24, 12–25 (2016).

    Article  PubMed  Google Scholar 

  35. 35

    Bar-Even, A. et al. The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. Biochemistry 50, 4402–4410 (2011).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Tawfik, D. S. Accuracy-rate tradeoffs: How do enzymes meet demands of selectivity and catalytic efficiency? Curr. Opin. Chem. Biol. 21, 73–80 (2014).

    CAS  Article  PubMed  Google Scholar 

  37. 37

    Luisi, P. L. An open question on the origin of life: the first forms of metabolism. Chem. Biodivers. 9, 2635–2647 (2012).

    CAS  Article  PubMed  Google Scholar 

  38. 38

    Laurino, P. & Tawfik, D. S. Spontaneous emergence of S-adenosylmethionine and the evolution of methylation. Angew. Chem. Int. Ed. 56, 343–345 (2017).

    CAS  Article  Google Scholar 

  39. 39

    Ralser, M. The RNA world and the origin of metabolic enzymes. Biochem. Soc. Trans. 42, 985–988 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40

    Keller, M. A., Piedrafita, G. & Ralser, M. The widespread role of non-enzymatic reactions in cellular metabolism. Curr. Opin. Biotechnol. 34, 153–161 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Horowitz, N. H. On the evolution of biochemical syntheses. Proc. Natl Acad. Sci. USA 31, 153–157 (1945).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Itoh, A. et al. Application of capillary electrophoresis-mass spectrometry to synthetic in vitro glycolysis studies. Electrophoresis 25, 1996–2002 (2004).

    CAS  Article  PubMed  Google Scholar 

  43. 43

    Sies, H. Strategies of antioxidant defense. Eur. J. Biochem. 215, 213–219 (1993).

    CAS  Article  PubMed  Google Scholar 

  44. 44

    David, W., Kilburn, M. R., Martin, S., John, C. & Brasier, M. D. Microfossils of sulphur-metabolizing cells in 3.4-billion-year-old rocks of Western Australia. Nat. Geosci. 4, 698–702 (2011).

    Article  Google Scholar 

  45. 45

    Lill, R. & Kispal, G. Maturation of cellular Fe–S proteins: an essential function of mitochondria. Trends Biochem. Sci. 25, 352–356 (2000).

    CAS  Article  PubMed  Google Scholar 

  46. 46

    R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2015).

Download references


We thank G. Averill and T. Littmann for helping with experiments. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001134), the UK Medical Research Council (FC001134) and the Wellcome Trust (FC001134). M.R. is supported by a Wellcome Trust grant, RG 093735/Z/10/Z, and a European Research Council Starting Grant, 260809. M.A.K. is supported by an Erwin Schrödinger postdoctoral fellowship (FWF, Austria, J3341). D.K. is supported by an Ad Futura studentship (Slovene Scholarship Fund).

Author information




M.A.K. and M.R. designed the research. M.A.K., D.K. and S.A.H. performed the research. M.A.K. and M.R. wrote the first draft of the paper, and all authors contributed to finalizing the manuscript.

Corresponding author

Correspondence to Markus Ralser.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Tables 6–13, Supplementary Figures 1–7. (PDF 1019 kb)

Supplementary Table 1

Reaction rate data for controls, Fe(II), peroxydisulfate and peroxydisulfate/ferrous sulfide. (XLS 45 kb)

Supplementary Table 2

Metal dependency rate data. (XLS 30 kb)

Supplementary Table 3

Z-score data. (XLS 505 kb)

Supplementary Table 4

Complete reaction list. (XLS 39 kb)

Supplementary Table 5

Scavenger experiment reaction rate data. (XLS 91 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Keller, M., Kampjut, D., Harrison, S. et al. Sulfate radicals enable a non-enzymatic Krebs cycle precursor. Nat Ecol Evol 1, 0083 (2017).

Download citation

Further reading