1. Metals and chemiosmosis; from the mitochondrion back to LUCA (and beyond …)

2. H+H+ H+H+ H+H+ bc oxidase Complex I mitochondrial respiration ATPase Cplx II NADPH NADP + succ fumarate O 2 H 2 O 0 -400 +400 +800 +1200 E m (mV) NADPH/NADP+ H 2 O / O 2 succinate/fumarate  E h is what drives the chain  E h =  E m +59mV/n log([ox]/[red])  G = -nF  E m

3. PSII b6fb6f oxygenic photosynthesis PSI aerobic respiration bc oxidase Complex I RCII bc anoxygenic photosynthesis bc RCI H 2 ase bc oxidase Knallgas reaction H 2 ase ? ? methanogenesis denitrification NO- reductase bc NO 2 - red NO 3 - red anaerobic nitrate respiration NO 3 - red Formate dehydro Hetero- disulfide reductase sulphate reduction H 2 ase hmc prokaryotes can do much more than that

4. PSII b6fb6f oxygenic photosynthesis PSI aerobic respiration bc oxidase Complex I RCII bc anoxygenic photosynthesis bc RCI H 2 ase bc oxidase Knallgas reaction H 2 ase ? ? methanogenesis denitrification NO- reductase bc NO 2 - red NO 3 - red anaerobic nitrate respiration NO 3 - red Formate dehydro Hetero- disulfide reductase sulphate reduction H 2 ase hmc 0 -400 +400 +800 +1200 E m (mV) H 2 / 2H + acetate/CO 2 methane/CO 2 HS - / SO 4 2- NO 2 - / NO 3 - H 2 O / O 2 Fe 2+ / Fe 3+ arsenite/arsenate N 2 / NO 3 - N 2 O / NO succinate/fumarate lactate/pyruvate

5. PSII b6fb6f PSI bc oxidase Complex I RCII bc RCI H 2 ase bc oxidase H 2 ase ? ? NO- reductase bc NO 2 - red NO 3 - red Formate dehydro Hetero- disulfide reductase H 2 ase hmc 0 -400 +400 +800 +1200 E m (mV) H 2 / 2H + acetate/CO 2 methane/CO 2 HS - / SO 4 2- NO 2 - / NO 3 - H 2 O / O 2 Fe 2+ / Fe 3+ arsenite/arsenate N 2 / NO 3 - N 2 O / NO succinate/fumarate lactate/pyruvate ATPase H+H+

6. PSII b6fb6f PSI bc oxidase Complex I RCII bc RCI H 2 ase bc oxidase H 2 ase ? ? NO- reductase bc NO 2 - red NO 3 - red Formate dehydro Hetero- disulfide reductase H 2 ase hmc ATPase H+H+ there are many bioenergetic systems

7. there are many bioenergetic systems but there is only one bioenergetic principle H+H+ in out ATPase reductantoxidant

8. A brief interlude

9. by the way, what is bioenergetics? the textbooks say: energy conversion by either fermentation or chemiosmosis both of these are based on electrochemical disequilibria, that is, presence of reducing and oxidising substrates which are not in thermodynamic equilibrium the electrochemical disequilibria are converted into chemical disequilibria, mainly in the form of ATP/ADP ratios

10. by the way, what is bioenergetics? the textbooks say: energy conversion by either fermentation or chemiosmosis

11. by the way, what is bioenergetics? the textbooks say: energy conversion by either fermentation or chemiosmosis “In fermentation, the redox process occurs in the absence of exogenous electron acceptors. In respiration, on the other hand, molecular oxygen or some other exogenous electron acceptor serves as a terminal electron acceptor. In fermentation, ATP is produced by substrate-level phosphorylation. In this process, ATP is synthesized directly from an energy-rich intermediate… This is in contrast to oxidative phosphorylation, in which ATP is produced at the expense of a proton motive force. In fermentation, by contrast, the proton motive force is not involved” (Brock, Biology of microorganisms, 2009 Edition, page 122)

12. by the way, what is bioenergetics? the textbooks say: energy conversion by either fermentation or chemiosmosis “In fermentation, the redox process occurs in the absence of exogenous electron acceptors. In respiration, on the other hand, molecular oxygen or some other exogenous electron acceptor serves as a terminal electron acceptor. In fermentation, ATP is produced by substrate-level phosphorylation. In this process, ATP is synthesized directly from an energy-rich intermediate… This is in contrast to oxidative phosphorylation, in which ATP is produced at the expense of a proton motive force. In fermentation, by contrast, the proton motive force is not involved” (Brock, Biology of microorganisms, 2009 Edition, page 122) according to the first criterion, several mechanisms involving a chemiosmotic ATP synthase must be classified as fermentations, such as for example acetogenesis or methanogenesis (and they indeed are in the Brock and in other textbooks)

13. by the way, what is bioenergetics? the textbooks say: energy conversion by either fermentation or chemiosmosis “In fermentation, the redox process occurs in the absence of exogenous electron acceptors. In respiration, on the other hand, molecular oxygen or some other exogenous electron acceptor serves as a terminal electron acceptor. In fermentation, ATP is produced by substrate-level phosphorylation. In this process, ATP is synthesized directly from an energy-rich intermediate… This is in contrast to oxidative phosphorylation, in which ATP is produced at the expense of a proton motive force. In fermentation, by contrast, the proton motive force is not involved” (Brock, Biology of microorganisms, 2009 Edition, page 122) Thus, the only truly distinguishing criterion is whether a proton motive force is involved or not!

14. by the way, what is bioenergetics? the textbooks say: energy conversion by either fermentation or chemiosmosis applying this criterion, fermentation is a very rare (and extremely inefficient) energy conversion mechanism whereas chemiosmosis is ubiquitous in all life

15. by the way, what is bioenergetics? chemiosmosis is THE way life converts energy

16. there are many bioenergetic systems but there is only one bioenergetic principle H+H+ in out ATPase reductantoxidant back to the main topic

17. then why is there this multitude of chemiosmotic energy conserving systems?

18. Energy is the fundamental prerequisite for life to exist just remember  G =  H - T  S this rationalises the extraordinary diversity of prokaryotic energy conversion  Life takes whatever electrochemical disequilibrium is out there to harvest energy

19. 0 -400 +400 +800 +1200 E m (mV) H 2 / 2H + acetate/CO 2 methane/CO 2 HS - / SO 4 2- NO 2 - / NO 3 - H 2 O / O 2 Fe 2+ / Fe 3+ arsenite/arsenate N 2 / NO 3 - N 2 O / NO succinate/fumarate lactate/pyruvate mitochondria Thiocapsa, Aquifex, … (Knallgas reaction) Acidithiobacillus

20. the “zoo” of bioenergetic pathways

21. sulphate reduction denitrification Knallgas reaction nitrate respiration methanogenesis oxygenic photosynthesis thiosulphate respiration acetogenesis mixed acid fermentation DMSO respiration denitrification hydrocarbon reduction arsenite oxidation Fe 2+ oxidation anoxygenic photosynthesis arsenate respiration

22. Complex I formate dehydrogenase Photosystem 1 NO reductase cytochrome oxidase heterodisulphide reductase arsenite oxidase Cu-Nir cytcd 1 Nir RCII succinate dehydrogenase fumarate reductase arsenate reductase Sulphide:quinone oxidoreductase RCI nitrogenase ACS/CODH polysulphide reductase ethylbenzene dehydrogenase Photosystem 2 methylfuran reductase selenate reductaseFeFe-hydrogenase Rieske/cytb complex [NiFe]-dehydrogenase formate hydrogenlyase methylreductase this gets even worse when we look at bioenergetic enzymes

23. is studying the diversity of bioenergetic pathways like collecting stamps? fortunately bioenergetics is much less diverse than it might seem!

24. the Rieske/cytb complex (alias Complex III, bc 1 complex, b 6 f complex …) arsenite oxidase

25. the Rieske/cytb complex (alias Complex III, bc 1 complex, b 6 f complex …)

26. Cytochrome oxidase (alias Complex IV, aa3, SoxM …) N 2 O reductase

27. Cytochrome oxidase (alias Complex IV, aa3, SoxM …) N 2 O reductase

28. Polysulphide reductase periplasmic nitrate reductase (Nap)

29. Polysulphide reductase periplasmic nitrate reductase (Nap)

30. Complex I (peripheral arm) [NiFe] - H 2 ase

31. Complex I (peripheral arm) [NiFe] - H 2 ase

32. - in all these cases the parent enzymes are functionally unrelated - the remaining subunits differ significantly for all pairs the similarities of subunits therefore do not reflect diversification of pre-existing enzymes  bioenergetic enzymes are built according to the LEGO principle and the “zoo” of enzymes can be reduced to a few basic building blocks

33. Baymann et al. (2003) Phil.Trans. R. Soc. B 358, 267-274 “The redox protein construction kit”

34. Bioenergetic enzymes are strongly inter-related edifices  elucidate the evolutionary histories of enzymes and pathways  and eventually even deduce a likely evolutionary root of bioenergetics in general the project:

35. Bioenergetic enzymes are strongly inter-related edifices the method:  recognise protein superfamilies (building blocks) either via sequence or structure similarities  construct multiple sequence alignments (structure often allows to assess deeper evolutionary histories, e.g. photosynthetic reaction centres) (if 3D information is available, refine the alignment based on structural parameters see for example Lebrun et al. 2006 Mol.Biol.Evol. 23, 1180-1191 on the case of the Rieske protein ) (if you don’t have a structure, live with it but be aware that the reliability of the phylogenetic trees is determined by the quality of the alignment)  reconstruct phylogenetic trees (always be aware that phylogenetic trees are probabilities!!!!)  assess the validity of trees through phylogenetic marker traits (look for structure, function or sequence idiosyncrasies, e.g. see Ducluzeau et al. 2008 TiBS 34, 9-15 )  if possible, root the tree (using paralogs  protein superfamilies, e.g. see Duval et al. 2008 BMC Evol.Biol. 8:206 )

36. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  Distinguish between pre- and post-LUCA enzymes what the hell is LUCA? BACTERIA EUKARYA ARCHAEA origin of life « gene-pool era » Last Universal Common Ancestor LUCA

37. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  Distinguish between pre- and post-LUCA enzymes when was the age of LUCA ? a quick history lesson

42. Origin of life

43. Bacteria Archaea LUCA Last Universal Common Ancestor

44. Origin of life Bacteria Archaea LUCA anoxygenic photosynthesis oxygenic photosynthesis

45. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes how can we do that? easy! (if you believe a vast majority of articles) “[Class I and II RNRs] … existed before the divergence of archaea and eubacteria during evolution as today they are abundantly present in both domains of life” Nordlund and Reichard 2006 Annu. Rev.Biochem. 75, bottom right on page 700 Equivalent arguments for N 2 O reductase by Zumft and Kroneck for nitrogenase by Richardson etc etc etc This completely ignores the possibility of Horizontal Gene Transfer (HGT)

46. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes Archaea Bacteria mitochondrial cytochrome oxidase (a member of the SoxM-group of heme-copper oxidases)  perfect Archaea/Bacteria dichotomy  cytochrome oxidase (an O 2 reductase!) in LUCA  O 2 in the Archaean?

47. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes Archaea Bacteria Three enzyme families are paralogs of SoxM, that is, SoxB- and cbb 3 -type O 2 reductases as well as NO reductase

48. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes Archaea Bacteria Three enzyme families are paralogs of SoxM, that is, SoxB- and cbb 3 -type O 2 reductases as well as NO reductase SoxM SoxB c-NOR q-NOR cbb 3

49. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes Archaea Bacteria Three enzyme families are paralogs of SoxM, that is, SoxB- and cbb 3 -type O 2 reductases as well as NO reductase SoxM SoxB cbb 3 q-NOR c-NOR LUCA  SoxM appeared in Archaea and was later horizontally transferred into the Bacteria

50. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes post-LUCA another case: the Rieske/cytb complex

51. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes post-LUCA another case: the Rieske/cytb complex

52. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes pre-LUCA another case: the Rieske/cytb complex

53. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes another case: the Rieske/cytb complex pre-LUCA

54. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  distinguish between pre- and post-LUCA enzymes 2 criteria 1- root lies in between archaeal and bacterial domains 2- significant coincidence between protein and species trees

55. Complex I (peripheral arm) Bioenergetic enzymes are strongly inter-related edifices what can we learn?  detect enzymes (or subunits thereof) which emerge from some other enzyme [NiFe] H 2 ase Mo-di-pterin [FeFe] H 2 ase Baymann et al. (2003) Phil.Trans. R. Soc. B 358, 267-274

56. Complex I (peripheral arm) Bioenergetic enzymes are strongly inter-related edifices what can we learn?  detect enzymes (or subunits thereof) which emerge from some other enzyme [NiFe] H 2 ase Mo-di-pterin [FeFe] H 2 ase  Complex I emerged post-LUCA through a combination of elements from a number of different enzymes

57. Bioenergetic enzymes are strongly inter-related edifices what can we learn?  for eukaryotes: determine source (host or endosymbiont) of individual enzymes (this provides a wealth of information on the evolutionary history of the eukaryotic cell)

58. So which enzymes where present in LUCA and which ones came later? -basically all Cu-enzymes are suggested from their molecular phylogeny to emerge post-LUCA  copper not bioavailable prior to O 2 makes geochemical sense Anbar 2008 Science 322, 1481

59. So which enzymes where present in LUCA and which ones came later? -Cu-enzymes probably appeared at around 2-2.5 billion years ago copper- enzymes

60. So which enzymes where present in LUCA and which ones came later? copper- enzymes LUCA several Mo-di-pterin enzymes NO reductase [NiFe] H 2 ase ACS/CODH Enzymes such as hydrogenases formate hydrogenlyase or ACS/CODH had been anticipated as ancient catalytic entities

61. So which enzymes where present in LUCA and which ones came later? copper- enzymes LUCA several Mo-di-pterin enzymes NO reductase [NiFe] H 2 ase ACS/CODH a possible presence of NO reductase was surprising since it implied the availability of a strong oxidant prior to O 2

62. 0 -400 +400 +800 +1200 E m (mV) H 2 / 2H + acetate/CO 2 methane/CO 2 HS - / SO 4 2- NO 2 - / NO 3 - H 2 O / O 2 Fe 2+ / Fe 3+ arsenite/arsenate N 2 / NO 3 - N 2 O / NO succinate/fumarate lactate/pyruvate

63. So which enzymes where present in LUCA and which ones came later? copper- enzymes LUCA several Mo-di-pterin enzymes NO reductase [NiFe] H 2 ase ACS/CODH in extant species NO reductase functions in the denitrification chain. What about LUCA? nitrate reductase?

64. Polysulfide reductase nitrate reductase arsenite oxidase tetrathionate reductase phylogenetic tree of the Mo-di-pterin superfamily (as of 2008)

65. energy anabolism various different energy conserving pathways the nitrogen cycle on modern planet Earth

66. the nitrogen cycle on modern planet Earth

67. the nitrogen cycle in LUCA

68. abiotic reactions fix atmospheric N 2 to yield NO, nitrate and nitrite (through electrical discharge and heat)

69. abiotic reactions reduce nitrate and nitrite to yield ammonia (in the upper crust by H 2 diffusing up from the Earth’s mantle) “serpentenisation”

70.  the nitrogen cycle in the Archaean was mainly driven by abiotic reactions  the “raison d’être” of the biological reactions was ENERGY

71. So which enzymes where present in LUCA and which ones came later? copper- enzymes LUCA several Mo-di-pterin enzymes NO reductase [NiFe] H 2 ase ACS/CODH

72. So which enzymes where present in LUCA and which ones came later? copper- enzymes LUCA several Mo-di-pterin enzymes NO reductase [NiFe] H 2 ase ACS/CODH a truncated version of the denitrification chain operated in LUCA

73. So which enzymes where present in LUCA and which ones came later? let’s summarise the present picture

74. H 2 ase ? ? methanogenesis denitrification NO- reductase NO 2 - red NO 3 - red anaerobic nitrate respiration NO 3 - red Formate dehydro Hetero- disulfide reductase H+H+ in out ATP ase reductantoxidant R/b AsIII-ox arsenite oxidation NO 3 - red H 2 ase Rnf?acetogenesis reductase ? H 2 ase polysulphide, thiosulphate, tetrathionate respiration MK MetPh mixed-acid fermentation Formate dehydro H 2 ase HCOO - H+H+ CO 2 NO H2H2 H2H2 H2H2 As III HCOO - S-compounds NO 3 - donors acceptors [] H2H2

75. beyond LUCA? (i.e. towards the origin of life) Observations not in line with the heterotrophic models (i.e. the organic soup scenarios) Günter Wächtershäuser’s “pyrite-surface catalysis” model Mike Russell’s “alkaline hydrothermal vent” model compare what we see with the predictions of the models

76. beyond LUCA? so far, the observations are in line with the predictions of the alkaline hydrothermal vent model what does this model say?

77. Credit: & Deb Kelley Lost City CH 4 :H 2 1:10 Martin et al. 2008, Nature Microbiol Rev 6, 805

78. Exothermic serpentinization From W. Bach Main energy discharge at alkaline off-axis site Ocean

79. NO/HNO 3 S0S0 Crust Ocean  H + gives protonmotive force & pyrophosphate?? [ H + + 2HPO 4 2-  HP 2 O 7 3- + H 2 O ] MoS 4 2- WS 4 2- H 2 ase ? ? methanogenesis denitrification NO- reductase NO 2 - red NO 3 - red anaerobic nitrate respiration NO 3 - red Formate dehydro Hetero- disulfide reductase R/b AsIII-ox arsenite oxidation NO 3 - red H 2 ase Rnf?acetogenesis reductase ? H 2 ase polysulphide, thiosulphate, tetrathionate respiration MK MetPh mixed-acid fermentation Formate dehydro H 2 ase HCOO - H+H+ CO 2 NO H2H2 H2H2 H2H2 As III HCOO - S-compounds NO 3 - donors [] H2H2 The energy metabolism pathways predicted by molecular phylogeny to be ancient correspond mostly to those of the alkaline vent model

80. furthermore, this model provides a rationalisation why chemiosmosis is THE principle of energy conversion for all life

81. a proton motive force (directed towards the inside of the vesicles) is a natural ingredient of the alkaline vent model

82. this is only a first glimpse the major part of the work still needs to be done by you!