Biophysics of Metalloenzymes Topics and Themes: (Metallo-) Proteins and Enzymes in the Cell 1) 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 Excitation-Energy and Electron Transfer 6) Proton Transfer 7) 8) Metal centers in Photosynthesis and Water Oxidation Biological Hydrogen Catalysis 9) Metal Cofactors in Nitrogen Fixation 10) Carbon Oxide Conversion at Metal Sites 11) Molybdenum Enzymes 12) Oxygen Reactions 13) Metal Centers in Human Diseases 14) Bioinspired Materials 15) Biophysics of Metalloenzymes M. Haumann
Photosynthesis http://bioenergy.asu.edu/
Solar Age – Hydrogen Economy efficient sunlight fuel cell conversion O 2 systems never-ending „fuel“ resources water H 2 H 2 O „Green“ fuel for heating, powering of machines, electricity....
Hypothetical Ideal Conversion Machine
Coupling Photosynthesis and Hydrogenases Photosynthesis: Production of atmospheric oxygen (O 2 ), reducing power, biomass Hydrogenases: Biological hydrogen (H 2 ) production Understanding the mechanisms of biological enzymes may pave the road to future biomimetic and biotechnological systems to switch to renewable energy resources. (1) Natural systems (whole organisms, biological catalysts) (2) Tailored biological enzymes for desired function (3) Biomimetic devices (e.g. artificial photosynthesis) (4) Synthetic catalysts (5) Future applications
Systems for O 2 and H 2 e.g. green algae (Chlamydomonas) Photo- bioreactors 10 µm
Taylored Enzymes 2H + H 2 PSI / H 2 ase chimera hydrogenase modul light e - photosystem modul electron donor Ihara et al Photochem Photobiol 2006
Semi-Artificial Hybrid Systems electrode electrode ... in progress in ongoing (inter)national collaborative research initiatives
Supramolecular Chemistry „Hydrogenase“ „Photosystem“ e - light „Chlorophyll“ „Tyrosine linker“ „Manganese complex“ light https://www.hfpeurope.org/uploads/1103/1597/SOLARH_STYRING_TechDays05_051208.pdf
Cell Leaf Chloroplast Thylakoid membrane with proteins lumen of light reactions stroma Thylakoids Biophysics of Metalloenzymes M. Haumann SS2014
Photosynthetic Electron Transfer Chain ATP-Synthase ATP Photosystem I Photosystem II Cytochrome Complex NADPH O 2 + 4H + 2H 2 O
Conversion of Light into Chemical Energy Z-Scheme Diagram Photosystem II Photosystem I
Overall Reactions dark (CO 2 fixation) and light (photosystems) reactions Photosystem II
Photosystem II Subunit composition Reaction center (D1D2) Antenna proteins Manganese Complex
PSII Structure Since 2001 Latest structure 1.9 Å (2011) 0.3 mm Electron density Mn complex PSII crystal membrane Manganese Loll et al Nature 2005 Complex
Cofactor Arrangement Iron Quinones Cytochrome Carotenoids Phaeophytins Chlorophylls Tyrosines Manganese Complex
Cofactor Distances http://pubs.rsc.org/en/content/articlehtml/2009/cs/b802262n
Oxygen Evolution Pattern Clark O 2 electrode O 2 + 4e- -> 2H 2 O O 2 from isolated PSII protein under short light flashes 2H 2 O -> O 2 + 4e- damping due to „misses“
Water Oxidation Cycle 2 H 2 O + 4 photons O 2 + 4 protons + 4 electrons S-state cycle O 2 Kok et al. 1970 Manganese Complex
Manganese Complex Structure 1.9 Å resolution crystal structure: Mn/Ca positions and µO resolved Crystal structure: all Mn-O bonds ~2.2 Å => Mn(II) mostly due to X-ray photoreduction Generation of high-valent structure by in- silico reversion of “damage” Kamiya et al. Nature 2011 Crystal structure of high-valent Mn 4 Ca cofactor can not be obtained by conventional X-ray crystallography due to rapid photoreduction
Fast Mn reduction by X-rays Time-resolved X-ray absorption spectroscopy (XAS) reveals fast Mn reduction and defines irradiation period or X- ray dose for „safe“ measurements Under crystallography conditions, all Mn(III/IV) ions are reduced within <1 s to Mn(II) Haumann et al. 2005
XFEL Structure Suga et al, Nature 2015 1.95 Å resolution No radiation damage!
EPR on Mn Complex S 2 state Mn(IV) 3 (III) Cox et al, Acc Chem Res 2013
XAS on Mn Complex Changes in S-state cycle -interatomic distances at Mn complex Mn-O/N Mn-Mn/Ca -metal-metal distances 20K S 1 -structural changes in reaction cycle Freeze-quench experiments (20K) S 2 and time-resolved experiment (RT) give essentially similar structural S 3 parameters S 0 RT 20K RT Haumann et al. Biochemistry 2005 Haumann et al. Biochemistry 2005
LD-XAS nn -linear-dichroism XAS relying on oriented sample and polarized X-ray beam Orientation of met6al- metal vectors (and metal- ligand) relative to membrane plane 3D structural model of metal complex http://www.springerimages.com/Images/LifeSciences/1-10.1007_s11120-009-9473-8-4
Structural & Redox Changes S 0 IV +III III Y z , 2H + , O 2 ox Y z 2H 2 O Y z , H + S 4 III IV +III IV ox Y z IV IV +IV S 1 III ox Y z IV +IV IV S 3 IV Y z Y z , H + ox Y z S 2 III
Atomic Level Model of Mn Complex S 1 -state µ-OH Manganese deprotonation oxidation Mn Mn Mn µ-O bridge formation Mn Substrate water binding
Electron Transfer Laser-flash induced optical transients S 1 ->S 2 Absorption difference spectra S 2 ->S 3 S 1 ->S 2 S 0 ->S 1 S 2 ->S 3 Van Leeuwen, Photosynth Res 1993 ET half-time Mn-complex -> Tyr Z + S 0 -S 1 30 µs S 1 -S 2 100 µs S 3 ->S 0 S 2 -S 3 250 µs S 3 -S 0 1000 µs (O 2 ) Gerenczer, Biochemistry 2010
Time-Resolved XAS on Mn Redox Changes Time traces K-edge shifts due to Mn redox D F(t) for excitation at 6552 eV A 10% 0,1 0F 1F 2F 3F OXIDATION A 70 µs XANES -0,3 0,0 0,3 0,6 0,9 1,2 1 0 6540 6544 S 1 S 2 S 3 S 0 190 µs B -0,3 0,0 0,3 0,6 0,9 1,2 0 1.1 ms C REDUCTION 6540 6550 6560 6570 Energy / eV -3 0 3 6 9 12 B Kinetics reveals that Mn is oxidized 30 µs D 3-times in S-state cycle -0,3 0,0 0,3 0,6 0,9 1,2 Time [ms] Haumann et al. Science 2005
Kß Emission on Mn Redox Changes Kß line energy shifts in reaction cycle due to Mn redox and coordination changes Zaharieva et al. Biochemistry 2016
Calibration with Reference Compounds Mn reference compounds Zaharieva et al. Biochemistry 2016
Coordination Changes in S-state Cycle Kß XANES PSII model complexes Kß emission line and K-edge absorption shape changes suggest ligation changes at Mn ions in S-state cycle Zaharieva et al. Biochemistry 2016
Coordination Change during S2->S3 Several options for possible structural changes remain – more research required Zaharieva et al. Biochemistry 2016
XFEL results Kern et al. Nature 2018
Oxygen binding to manganese New oxygen species bound to Mn1 in S 3 Kern et al. Nature 2018
Proton Release During Water Oxidation Absorption changes of pH-indicator Lumen pH- indicator strong pH-buffer (+inhibitors) Haumann & Junge Biochemistry 1994 Rapid proton release t 1/2 ~ 20 µs on all S-transitions Slow (1 ms) protons on S3->S0 oxygen evolving step in parallel to O 2 formation
pH-Dependent Changes in Stoichiometry -Rapid (~20 µs) proton release on all S-transitions -pH dependent amplitudes -protons are released when tyrosine Z is still oxidized (prior to electron transfer from Mn complex to Y Z +) -electrostatic origin of protons from amino acid side chains in response to positive charges on Mn causing pK shifts (Bohr-effect)
PT and ET monitored by XAS Mn reduction on S3-S0 oxygen-evolving step 6556 eV F max -F(t) 1 250 µs Kinetic deviation from single- 0,1 exponential behaviour (lag phase of -1 0 1 2 3 4 5 ~250 µs) indicates intermediate Time [ms] formation prior to Mn reduction Y Z Y Z Y Z + 0 + 200 µs 1.1 ms Intermediate S 3 S 0 State < 1 µs Mn reduction Neither Mn oxidation and O 2 -formation nor reduction
Kinetic H/D Isotope Effects Zaharieva, Dau, Haumann, Biochemistry 2016
Protons in PSII studied by PBD activation energy kinetic traces, f(T) H/D isotope effect Large H/D effect, large Ea, large pH-dependence have unraveled new proton release associated kinetic phases Klauss, Haumann, Dau, PNAS 2013
Reaction Sequence of S2->S3 Transition Spatio-temporal orchestration of electron and proton transfer essential for efficient water oxidation
Reaction Sequence of O 2 Release Step D1-Tyr Z D1-His190 CP43-Arg357 D1-Asp61 lumen Dau & Haumann Coord Chem Rev 2008
Protons at Acceptor Side Gated ET following coordination change at iron due to additional change on quinone Chernev et al. 2011
Alternating ET and PT Klauss, Haumann, Dau, PNAS 2013
Water Oxidation Under Pressure Hypothesis: O 2 product inhibition of water oxidation limits atmospheric O 2 level to ~20 % S 4 G No pO 2 limitation of 10 bar water oxidation! 2 bar S 2 p O 2 * -OOH (secondary effects 60 meV on Mn redox state) 0.2 bar S 0 +H + +O 2 XAS at high pO 2 Haumann et al, PNAS 2008
Cofactor Redox Potentials Zaharieva & Dau 2009
Energetics of Water Oxidation Zaharieva & Dau 2009
PSII Efficiency Zaharieva & Dau 2009
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