SLIDE 1
Ferritin Superfamily
Core 4-helix bundle Metal-binding motif
- C. trachomatis R2c (magenta),
- M. tuberculosis R2lox (blue)
Biophysics of Metalloenzymes Topics and Themes: 1) (Metallo-) - - PowerPoint PPT Presentation
Biophysics of Metalloenzymes Topics and Themes: 1) (Metallo-) - - PowerPoint PPT Presentation
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)
SLIDE 2
SLIDE 3
Andrews, Biochimica et Biophysica Acta 1800 (2010) 691–705
Member Groups
SLIDE 4
Conserved Metal Ligands
SLIDE 5
Diverse Reactivity
Krebs et al. Curr Opin Chem Biol. 2011; 15(2): 291–303.
SLIDE 6
Ferritin: Metal Transport and Storage
www.uni-ulm.de/fileadmin/website_uni_ulm/presse/pressemitteilungen/2013/Ferritin.jpg
Ferritin
http://www.jbc.org/content/286/29/25620/F1.large.jpg
SLIDE 7
Ribonucleotide Reductase
ß2 Deoxyribonucleotide synthesis
SLIDE 8
Biophysics of Metalloenzymes
- M. Haumann
SS2014
Radical mechanism
Deoxyribonucleotide formation
SLIDE 9
Three Types of RNRs
FeFe MnMn MnFe
SLIDE 10
MnFe, FeFe, or MnMn
Anomalous diffraction
SLIDE 11
Features
Cotruvo & Stubbe, Annu. Rev. Biochem. 2011. 80:733– 67
SLIDE 12
Tyrosine as Cofactor
- E. coli
R2-Ia RNR
- C. trachomatis
R2-Ic RNR
Jiang et al., Biochemistry. 2008, 47(52): 13736–13744. Gräslund, Annu. Rev. Biophys.
- Biomol. Struct. 1996. 25t259-86
X-band EPR
SLIDE 13
Tyrosine Radical
Bennati et al., Biol. Chem. 386, 1007–1022, 2005
Reorientation of reduced tyrosine Y122 (cyan) as compared with the radical form (green, g- tensor axes yellow), showing a disruption of the radical from the network of hydrogen-bonded amino acids in R2. Angular-dependent 94-GHz EPR spectra of the tyrosyl radical Y122• in R2 single crystals of E. coli RNR.
SLIDE 14
EPR Studies
Mn(III)Fe(III) Fe(III)Fe(III) Fe(IV)
Leidel et al., Biochimica et Biophysica Acta 1817 (2012) 430–444
Mn(IV)Fe(IV)
SLIDE 15
ET between a and ß subunits
SLIDE 16
Crystal Structures
SLIDE 17
Rapid X-ray Photoreduction
Sigfridsson et al. J Biol Chem 2013, 288(14):9648-61
FeFe site in Ct R2 MnFe site in Ct R2
SLIDE 18
Structural Changes upon Redox
DFT
Sigfridsson et al. J Biol Chem 2013, 288(14):9648-61
SLIDE 19
Free Electron Laser
X-ray femtosecond pulses
Structures of high-valent metal sites without X-ray photoreduction Scattering faster than ET from radicals „Measure before destroy“ approach
SLIDE 20
Cofactor Formation
SLIDE 21
Assembly
Griese et al. JBIC 2014
SLIDE 22
Ligand-Binding Oxidase
Griese et al., PNAS 2013, 110, 17189–17194
fatty acid ligand crosslink
SLIDE 23
Structural Comparison
- Fig. 7 Structural comparison of R2c and R2lox. a Superposition of the reduced Mn/Fe-bound
states of R2lox (4HR4 [28]) and R2c (4M1I [56]). b Superposition of the oxidized Mn/Fe-bound state of R2lox (4HR0 [28]) and the oxidized Fe/Fe-bound state of R2c (SYY [51]). Amino acid residues of R2lox are shown in cyan, the fatty acid ligand in blue, and Mn and Fe as purple and orange spheres, respectively, while R2c is shown in gray
Griese et al. JBIC 2014
SLIDE 24
Protonation of Ligands
SLIDE 25
NRVS and QM/MM Studies
Kositzki et al. 2016
SLIDE 26
Specific Cofactor Structure and H-Bonding
SLIDE 27
Biophysics of Metalloenzymes
- M. Haumann
SS2014
Reactivity
Griese et al., PNAS 2013, 110, 17189–17194
Hypothetical desaturation reaction (substrate) Amino acid crosslink formation
SLIDE 28
Reaction Cycles
Reece & Nocera. Annu Rev Biochem 2009 Bollinger et al. Current Opinion in Structural Biology 2008, 18:650–657
SLIDE 29
Intermediate X
SLIDE 30
O2 Cleavage Energetics
Griese et al. JBIC 2014
SLIDE 31
Long-Range PCET
http://web.mit.edu/biochemistry/research.htm
SLIDE 32
Methane – Liquid Fuel (Methanol)
C-H bond activation O2 activation
SLIDE 33
Technical Methanol Synthesis
High energy demand Low yield Low selectivity Many byproducts
SLIDE 34
Methane Monooxygenase (MMO)
Diiron active site structures of (a) McMMOHox (PDB ID 1MTY) and (b) Mc MMOHred (PDB ID 1FYZ).
Tinberg & Lippard, ACCOUNTS OF CHEMICAL RESEARCH, 280–288, 2011, 44(4)
Top-10 challenges in catalysis
SLIDE 35
Catalytic Cycle
Tinberg & Lippard, ACCOUNTS OF CHEMICAL RESEARCH, 280–288, 2011, 44(4)
SLIDE 36
Biophysics of Metalloenzymes
- M. Haumann
SS2014
Peroxo Intermediate
Tinberg & Lippard, ACCOUNTS OF CHEMICAL RESEARCH, 280–288, 2011, 44(4)
SLIDE 37
Biophysics of Metalloenzymes
- M. Haumann
SS2014
Intermediate Q
3 proposed pathways 4 proposed structures
Tinberg & Lippard, ACCOUNTS OF CHEMICAL RESEARCH, 280–288, 2011, 44(4)
SLIDE 38
Summary
Ferritin superfamily Members & reactivities Ferritin iron storage Ribonucleotide reductase Deoxyribonucleotide formation 3 types of RNR (FeFe, MnFe, MnMn) Tyrosine radical Crystal structures X-ray photoreduction Codactor assembly Ligand binding oxidase Amino acid crosslink Reaction cycles O2 cleavage PCET Methane to methanol Methane monooxygenase
SLIDE 39
Literature
Högbom, Metal use in ribonucleotide reductase R2, di-iron, di-manganese and Heterodinuclear, Metallomics 3, 2011, 93–216 Tinberg & Lippard, Dioxygen Activation in Soluble Methane Monooxygenase, ACCOUNTS OF CHEMICAL RESEARCH 44, 2011, 280–288 Reece & Nocera, Proton-Coupled Electron Transfer in Biology, Annu. Rev. Biochem.
- 2009. 78:673–99