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) 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

Ferritin Superfamily Ferritin Metal-binding motif Various other enzymes (purple acid phosphatase…) Methane Monooxygenases Ribunucleotide Reductases Ligand-binding oxidases (e.g. in human parasites) Core 4-helix bundle C. trachomatis R2c (magenta), M. tuberculosis R2lox (blue) and Prochlorococcus marinus alkane synthesizing protein (PDB id: 2oc5) (green).

Member Groups Andrews, Biochimica et Biophysica Acta 1800 (2010) 691 – 705

Conserved Metal Ligands

Diverse Reactivity Krebs et al. Curr Opin Chem Biol. 2011; 15(2): 291 – 303.

Ferritin: Metal Transport and Storage Ferritin www.uni-ulm.de/fileadmin/website_uni_ulm/presse/pressemitteilungen/2013/Ferritin.jpg http://www.jbc.org/content/286/29/25620/F1.large.jpg

Ribonucleotide Reductase Deoxyribonucleotide synthesis ß 2

Deoxyribonucleotide formation Radical mechanism Biophysics of Metalloenzymes M. Haumann SS2014

Three Types of RNRs FeFe MnMn MnFe

MnFe, FeFe, or MnMn Anomalous diffraction

Features Cotruvo & Stubbe, Annu. Rev. Biochem. 2011. 80:733 – 67

Tyrosine as Cofactor X-band EPR E. coli R2-Ia RNR Gräslund, Annu. Rev. Biophys. Biomol. Struct. 1996. 25t259-86 C. trachomatis R2-Ic RNR Jiang et al., Biochemistry . 2008, 47(52): 13736 – 13744.

Tyrosine Radical Angular-dependent 94-GHz EPR spectra of the tyrosyl radical Y122• in R2 single crystals of E. coli RNR. 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. Bennati et al., Biol. Chem. 386, 1007 – 1022, 2005

EPR Studies Mn(III)Fe(III) Fe(III)Fe(III) Fe(IV) Mn(IV)Fe(IV) Leidel et al., Biochimica et Biophysica Acta 1817 (2012) 430 – 444

ET between a and ß subunits

Crystal Structures

Rapid X-ray Photoreduction FeFe site in Ct R2 MnFe site in Ct R2 Sigfridsson et al. J Biol Chem 2013, 288(14):9648-61

Structural Changes upon Redox DFT Sigfridsson et al. J Biol Chem 2013, 288(14):9648-61

Free Electron Laser Structures of high-valent metal sites without X-ray photoreduction Scattering faster than ET from radicals X-ray femtosecond pulses „Measure before destroy“ approach

Cofactor Formation

Assembly Griese et al. JBIC 2014

Ligand-Binding Oxidase fatty acid ligand crosslink Griese et al., PNAS 2013, 110, 17189 – 17194

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

Protonation of Ligands

NRVS and QM/MM Studies Kositzki et al. 2016

Specific Cofactor Structure and H-Bonding

Reactivity Amino acid crosslink formation Griese et al., PNAS 2013, 110, 17189 – 17194 Hypothetical desaturation reaction (substrate) Biophysics of Metalloenzymes M. Haumann SS2014

Reaction Cycles Reece & Nocera. Annu Rev Biochem 2009 Bollinger et al. Current Opinion in Structural Biology 2008, 18:650 – 657

Intermediate X

O 2 Cleavage Energetics Griese et al. JBIC 2014

Long-Range PCET http://web.mit.edu/biochemistry/research.htm

Methane – Liquid Fuel (Methanol) C-H bond activation O 2 activation

Technical Methanol Synthesis High energy demand Low yield Low selectivity Many byproducts

Methane Monooxygenase (MMO) Top-10 challenges in catalysis 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)

Catalytic Cycle Tinberg & Lippard, ACCOUNTS OF CHEMICAL RESEARCH, 280 – 288, 2011, 44(4)

Peroxo Intermediate Tinberg & Lippard, ACCOUNTS OF CHEMICAL RESEARCH, 280 – 288, 2011, 44(4) Biophysics of Metalloenzymes M. Haumann SS2014

Intermediate Q Tinberg & Lippard, ACCOUNTS OF CHEMICAL RESEARCH, 280 – 288, 2011, 44(4) 4 proposed structures 3 proposed pathways Biophysics of Metalloenzymes M. Haumann SS2014

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 O 2 cleavage PCET Methane to methanol Methane monooxygenase

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 Andrews, The Ferritin-like superfamily: Evolution of the biological iron storeman from a rubrerythrin-like ancestor, Biochimica et Biophysica Acta 1800 (2010) 691 – 705 Cotruvo & Stubbe, Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo, Annu. Rev. Biochem. 2011. 80:733 – 67 Stubbe & Cotruvo, Control of metallation and active cofactor assembly in the class Ia and Ib ribonucleotide reductases: diiron or dimanganese? Curr Opin Chem Biol. 2011, 15(2): 284 – 290 Stubbe et al., Radical Initiation in the Class I Ribonucleotide Reductase: Long-Range Proton-Coupled Electron Transfer? Chem. Rev. 2003, 103, 2167-2201 Griese et al., Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein, PNAS, 2013, 110, 17189 – 17194 Leidel et al., High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases, Biochimica et Biophysica Acta 1817 (2012) 430 – 444 Sigfridsson et al., Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase, J Biol Chem. 2013, 288(14):9648-61