Biophysics of Metalloenzymes Topics and Themes: 1) (Metallo-) Proteins and Enzymes in the Cell 2) Some Principles of Coordination Chemistry 3) Methods for Investigation at Molecular Level 4) Overview on Metal Cofactors in Biology 5) Cofactor Assembly and Maturation 6) Excitation-Energy and Electron Transfer 7) Proton Transfer 8) Metal centers in Photosynthesis and Water Oxidation 9) Biological Hydrogen Catalysis 10) Metal Cofactors in Nitrogen Fixation 11) Carbon Oxide Conversion at Metal Sites 12) Molybdenum Enzymes 13) Oxygen Reactions 14) Metal Centers in Human Diseases 15) Bioinspired Materials
Hydrogenase reaction 2H + + 2e - H 2 Enzymes: >10 4 s -1
Hydrogen for the future Belgian architect Vincent Callebaut has designed a conceptual transport system that would involve airships powered by seaweed. Called Hydrogenase, the project envisages that by 2030 there could be farms in the ocean producing biofuel from seaweed and acting as hubs for the aircraft. http://www.dezeen.com/2010/05/07/hydrogenase-by-vincent-callebaut/
Carbon-free energy Hydrogen production and cleavage cycle http://www2.hu-berlin.de/biologie/microbio/application/sm_application.htm
Hydrogenase fuel cell Usage of 3 % H 2 below combustion limit in air by NiFe hydrogenase can drive an electrical device for >24 h http://www2.hu-berlin.de/biologie/microbio/olenz/Oliver_Lenz_Research.html http://www.rsc.org/Publishing/ChemScience/Volume/2007/01/enzyme_fuel_cells.asp
Hydrogen producing organisms / evolution Bacteria (Desulfovibrio, Clostridium, Rhodobacter, Knallgas, archaea, methanogens…) The three types of hydrogenases Green algae (Chlamydomonas…) (NiFe, FeFe, Fe) have evolved independently and convergently Phylogenetic tree of NiFe FeFe hydrogenase diversification hydrogenase large subunit Vignais, FEMS Microbiol Rev 25 (2001) 455
H 2 producers in soil Hydrogen producing bacteria in soil
Types of hydrogenases Iron-only containing hydrogenases [FeFe]-hydrogenase H-cluster [4Fe4S] [2Fe2S] H-cluster H-cluster ([4Fe4S]) [4Fe4S] Chlamydomonas reinhardtii (2LX4) Desulfovibrio desulfuricans (1HFE) Chlostridium pasteurianum (3C8Y) [Fe]-hydrogenase Fe-guanylyl-pyridone-cofactor Methanocaldococcus jannaschii (3F47)
Hydrogenases ff [NiFe]-hydrogenase [NiFeSe]-hydrogenase PH MBH NiFe-cofactor NiFeSe-cofactor [4Fe4S] [4Fe3S] [3Fe4S] [4Fe4S] Ralstonia eutropha (3RGW) Desulfovibrio gigas (1WUK) Desulfomicrobium baculatum (1CC1) soluble (SH) membrane bound (MBH)
H 2 sensors Dimeric Linker Protein- hydrogenase domain histidine kinase PAS domain Shafaat BBA 2013 H 2 sensing triggers expression of energy converting hydrogenases
Iron-sulfur clusters in H 2 ases [4Fe4S] His Cys [2Fe2S] [3Fe4S] [4Fe3S2O] Glu [4Fe3S] ox red Pb Biophysics of Metalloenzymes M. Haumann SS2014
Active sites Iron-only hydrogenases [Fe]-hydrogenase M.j. wt (3F47) M.j. C176A+DTT (3F46) Cys [FeFe]-hydrogenase C.p. ox (3C8Y) D.d. red (1HFE) C.p. +CO (1C4C) Cys Cys H-cluster Cys Cys
Structure of H-cluster in FeFe-hydrogenases [4Fe4S] H cys cys adt cys cys Fe p Fe d [2Fe] H
Crystal structures of the H-cluster Bridging ligand long time debated bacterial enzymes apo-HydA1 (green algae)
Maturation of FeFe-hydrogenase 3 maturases sufficient to get active enzyme in vivo and in vitro Similar H- cluster on HydF maturase and HydA enzyme
In vitro maturation Apo-protein, maturases and inorganic compounds produce active enzyme http://openi.nlm.nih.gov/detailedresult.php?img=3105041_pone.0020346.g001&req=4
In vitro maturation 2 with maturase HydF without maturases Happe et al. 2013-2016
Reconstitution with chemical compounds Esselborn et al. Nat Chem Biol. 2013, 9:607-9.
In vitro maturation 3 Spontaneous activation of [FeFe]- hydrogenase in vitro with or without maturase HydF and inorganic synthetic cofactors. Works for HydA1 and CpI! Happe et al. 2013-2016
Modified cofactors from in vitro maturation Infrared control of non-natural synthetic cofactor insertion Siebel et al. Biochemistry 2015
Chalcogenide substitution In vitro maturation with S/Se exchanged synthetic cofactors => full activity! Noth et al. Angew. Chem. 2016
Maturation of FeFe-hydrogenase All CO and CN ligands are derived from tyrosine Kuchenreuther PLOSone 2011
Active sites of NiFe-hydrogenases [NiFeSe]-hydrogenase D.b. H 2 -red (4KN9) D.v. air-ox (2WPN) D.b. air-ox (4KL8) Sec Cys Cys Cys [NIFe]-hydrogenase D.f. H 2 -red (1YRQ) D.v. +CO (1UBC) D.v. ox Ni-A (1WUI) D.v. ox Ni-B (1WUJ) Cys Cys Cys Cys R.e. H 2 -red (3RGW) H.m. air-ox (3AYZ) E.c. fecy-ox (3USC)
Assembly and maturation of an MBH ( R. eutropha ) Synthesis of the Fe(CN) 2 (CO) site Carbamoyl phosphate oligomerization translocation Ni insertion
Oxygen tolerance in NiFe-H 2 ases O 2 tolerant membrane-bound enzyme (R. eutropha) Reactivation is fast after O 2 removal, residual activity at ambient pO 2 Standard O 2 sensitive periplasmic enzyme (D. gigas) Reactivation requires hours at low potentials. No activity at ambient pO2 Vincent et al. Dalton Trans.,2005, 3397-3403
Electrochemical experiments Cyclic voltametry Rotating graphite disk electrode H 2 production H 2 cleavage inactivation Thermodynamic reversible H 2 reactions! H 2 /H+ equilibrium potential Almost no overpotential
EPR results NiFe site FeS clusters EPR monitors Ni(III) or Ni(I) states, Ni(II) is EPR silent 61 Ni Different FeS clusters in O 2 tolerant and sensitive enzymes g-values in various species Lubitz Chem Rev 2014 Biophysics of Metalloenzymes M. Haumann SS2014
FTIR fingerprinting Spectro-electrochemistry Lubitz et al, Chem Rev 2014
SEIRAS
A new FeS cluster in MBHs EXAFS reveals different FeS clusters in PH O 2 tolerant MBHs S Cys20 S S Cys148 B Fe Fe S I Cys112 S 60 Fe S 10 Fe S 3 EXAFS xk Cys17 S FT of EXAFS II MBH 0 S S Cys20 Cys120 S S 3 6 9 12 Cys149 Fe -1 k / Å S Fe d Cys115 S S Fe S Cys19 0 Fe S 1 2 3 4 5 reduced distance / Å S Cys17 Two additional cysteines in small subunit, Fritsch, Biochemistry 2012 cluster coordinated by 6 cys Sigfridsson, BBA 2014
Structural changes Proximal cluster [4Fe3S] Two oxidizing transitions in [4Fe3S] vs only one in [4Fe4S]
States of NiFe(Se) H 2 ases Shafaat, BBA 2013 Biophysics of Metalloenzymes M. Haumann SS2014
NiFe activation and inactivation Lubitz, Chem Rev 2014
Modifications at active site Cys597 PH MBH Thr553 Cys530 Ile531 Leu598 Ser486 (Met) Arg530 O Arg463 Ala599 c Ala532 Cys75 Cys65 O b Gly76 Gly66 Cys600 Cys533 a a Val77 Val67 Cys78 Cys68 Thr79 Thr69 Gly80 Tyr70 (Cys, Gly) (Thr, Leu) Cys81 Val71 His82 His72 (Ile, Val) (Thr) Ni-A (PH) O 2 , n H + OH n - O b OH n H O aerobic OH - , (FeS) n - (FeS) Ni 2+ Fe 2+ Ni 3+ Fe 2+ anoxic O c OH n Sigfridsson, BBA 2014 OH n , n Ox n Red Ni-B (MBH)
Catalytic cycle in [FeFe]-hydrogenase Lubitz, Chem Rev 2014
Site-selective XAS/XES on the H-cluster [4Fe4S] apo-HydA1 A [4Fe4S] apo-HydA1 7045 eV [2Fe] adt cys [4Fe4S] apo-HydA1 [2Fe] adt 7060 eV cys normalized XANES (Kß) 1 c2v * Kß 1,3 normalized Kß emission cys [4Fe4S] apo-HydA1 [2Fe] adt cys [2Fe] adt 0 Kß ´ 7045 7060 emission energy / eV 7110 7120 7130 7140 7150 excitation energy / eV C B Kß 2,5 high-spin d 5 Fe(III) low-spin d 7 Fe(I) [4Fe4S] [2Fe] Kß emission 3 d 3 p [4Fe4S] apo-HydA1 v2c Kß 1,3 Kß ´ Kß 1,3 [2Fe] adt 1 s 7080 7095 7110 emission energy / eV resonant excitation Chernev Inorg Chem 2014
XAS/XES on red and sred states core-to-valence transitions valence-to-core transitions [4Fe4S] H A B ([4Fe4S]-[2Fe]) H 4 0,5 red sred Comparison of red experimental and DFT exp sred calculated spectra DFT reveals good exp ( sred – red ) exp DFT agreement for a pre-edge absorption bridging hydride in the DFT ( sred – red ) x3 2,5 emission red super-reduced state [2Fe] H sred exp DFT exp [4Fe4S] H Kß red sred red DFT DFT sred Fe d Fe p DFT [2Fe] H red DFT Fe d Fe p DFT sred Hy DFT red 0,0 exp DFT DFT sred Hy 0 ( sred – red ) 7090 7110 7111 7114 emission energy / eV excitation energy / eV Chernev Inorg Chem 2014
Briding hydride in sred H + [4Fe4S] [H + ] cys I I CO NC OC CN e - H + sred H 2 H H 2+ 2+ [4Fe4S] [4Fe4S] [H + ] cys cys I I II I CO CO NC e - H + NC C C OC OC CN CN O O red ox Chernev Inorg Chem 2014
Calculation of IR modes by DFT IR spectrum of H ox -CO
Reversible 13 CO labelling H ox H ox -CO
DFT on IR frequencies of CO and CN „standard“ H ox 2000 apical open site at Fe d -1 calculated CO band frequency / cm H ox -CO 1950 apical CO at Fe d apical CN at Fe d CO: 1900 p m d1 d2 12121212 13131313 „rotamers“ 1850 12131212 12121312 12121213 12131312 1800 12131213 12121313 12131313 1750 1750 1800 1850 1900 1950 2000 -1 experimental CO band frequency / cm CO/CN geometry in H-cluster may not be settled
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