Bioinorganic Chemistry & Biophysics of Plants

Bioinorganic Chemistry & Biophysics of Plants University of Konstanz, SS 2012 [email protected]

1 Bioinorganic Chemistry & Biophysics of Plants Lectures P. Kroneck

1. Basics of Coordination Chemistry in Biological Systems (transition metals – general properties – structural/functional aspects)

2. Magnetic Resonance Spectroscopy as Structural Tool (electron paramagnetic resonance, nuclear magnetic resonance)

3. Global Cycles of the Elements (nitrogen, sulfur, metals, and metalloenzymes)

4. From Physiology to Biochemistry to Spectroscopy and Protein Structure (copper and iron proteins)

5. How to give a good seminar & how to prepare a good poster

2 "Bioinorganic Chemistry & Biophysics of Plants" Introductory Books & References

Frausto da Silva, Williams, 2001 The biological chemistry of the elements, Oxford University Press

H. B. Gray, E. I. Stiefel, J. Selverstone Valentine, I. Bertini, 2007 Biological Inorganic Chemistry: Structure and Reactivity, University Science Books

R. R. Crichton, 2012 Biological Inorganic Chemistry, 2nd edition, Elsevier

Messerschmidt, Huber, Poulos, Wieghardt (eds), 2001; 2004; 2009 on-line Handbook of Metalloproteins, John Wiley & Sons, LTD

Chemical Reviews, 1996; 2004 Special Issues on Bioinorganic Enzymology, 96, 2237; 104, 347

3 Useful web sites http://http://www.ebi.ac.uk/pdbe/ comprehensive database of all published protein structures http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi comprehensive databases (genomes, genes, proteins, inherited diseases...etc) and various search tools http://www.brenda-enzymes.org/ comprehensive enzyme database including information on metal requirements http://www.webelements.com periodic table of the elements including useful information on each element

4 PDB and PROMISE Databases

• PDB = Protein Data Bank: http://www.rcsb.org/pdb/home.do; type in the search field the PDB number: • 1A70 (for Ferredoxin), 3SBP for Nitrous Oxide Reductase • PROMISE at Scripps, San Diego: htpp://metallo.scripps.edu/PROMISE/

5 Bringing Inorganic Chemistry to Life... Biochemistry Biogeochemistry Physiology Molecular Biology Biophysics Bioinorganic Chemistry Microbiology Environmental Spectroscopy Chemistry

Structural Biology Catalysis Physical Chemistry Medical Chemistry/Toxicology 6

Part 1: Basics of Coordination Chemistry in Biological Systems (transition metals – general properties – structural and functional aspects)

“The application of metals to treat human ailments dates back at least to the fifth century B.C., and the study and mimicry of metals in biology is a vibrant subject today”…. Stephen Lippard, J Am Chem Soc (2010), 132, 141689-14693

7 The elements/metals of life H He www.webelements.com

Li Be B C N O F Ne

Na Mg Al Si P S Cl Ar

K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr

Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe

Cs Ba La Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn

Essential Transition metals (ions) are Ca: 1.2 kg Transition Metals under strict control within the K: 150 g Fe: 4.5 g living cell; usually bound to Na: 70 g Zn: 2.3 g proteins, peptides, or other Mg: 20-30 g Cu: 72 mg molecules. Mo: 9 mg In CHEMISTRY we call this a TRANSITION METAL COMPLEX 8 Why Investigate Metals in Biology ? • There is hardly any important process in nature which does not depend on a metal ion; ~ 1/3 of the proteins of the human genome depend on metal ions • Novel Materials, Structures and Reactions • Trigger - Signaling - Sensing - Regulation • Acid-Base Catalysis • Redox – Proton & Electron Transfer (coupled, conservation of energy) 9 Case 1:Metals in Medicine - DNA – cis-platin

10 Case 2: Nitrogen Fixation - Nitrogenase http://en.wikipedia.org/wiki/Nitrogen_cycle

11 Case 3: Respiration – Reduction of O2 to H2O Synthesis of ATP – proton-coupled electron transfer (PCET)

12 Goal: From highly resolved 3D & Electronic Structure to Function to Mechanism

ABS

)

- M

m 2

(



0 25000 20000 15000 10000 Wavenumber (cm-1) EPR

2800 3000 3200 3400 Magnetic field (G)

13 Why (Transition)Metal Ions ?

• Positively Charged • Open Shell Systems – Lewis Acids – No Problems with Spin Restriction – Stabilization of Anions • Sterochemically Flexible • Loosely Bound Electrons – Large Variety of – Redox Active Structures. – Multiple Redox States – Little Reorganization – Easily tunable Redox – Facile Ligand Addition/Dissociation Potential • Facilitate Reactions of • Coupled Redox/Acid Base Bound Ligands Chemistry

14 (Bio)Metals – Perodic Table - Electrons

H www.webelements.com He Li Be B C N O F Ne Na Mg Al Si P S Cl Ar K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr Mo W

 3d i 4s 3p 3s

2p 2s

1s 15 3d-Orbitals

d 2 2 y z r

x z z

y d yz d xz y x x

z z

y y d 2 2 d x  y xy x x

16 Basic Features of a Metal Protein Complex Chem. Rev. 1996, 96, 2239-2314 (1996) RH Holm, P Kennepohl, E I Solomon, Structural and Functional Aspects of Metal Sites in Biology

Prot-L| ------M d- d+ strong attraction

- + CH4 + O2 +2e +2H  H3COH + H2O

Chemistry at the Catalytic Center (Active site) of the Iron Enzyme Methane Monooxygenase

17 Protein Ligands – Amino Acid Residues N O S

Tyr

His Cys

Glu(+Asp)

Met Lys Ser 18 Tetrapyrrole - Versatile Ligand in Biology

O2 Respiration Photosynthesis O O O

O O O O A B O NH HN (Bacterio)- NH HN Chlorophylls (Mg+2) +2 D C Hemes (Fe ) O O O O

O O O O

Sirohemes VitB (Co+2) +2 12 (Fe , +2 F430 (Ni ) Fe-S cluster)

N & S Cycles

Met biosynthesis 19 Methanogenesis Molybdopterin, a Dithiolene Ligand (binds both Mo and W) JOURNAL of BIOLOGICAL CHEMISTRY (2009) Vol. 284, p. e10, N Kresge, R D Simoni, R L Hill: The Discovery and Characterization of Molybdopterin - the Work of K. V. Rajagopalan

Mo-S-Cu Cluster in CO Dehydrogenase from Oligotropha carboxidovorans : + - CO + H2O → CO2 + 2 H + 2e H Dobbek et al., Proceedings National Academy of Sciences/USA, 99, 15971-15976 ( 2002) 20 Geometry – Coordination Number

3 Trigonal Trigonal pyramidal T-shape 4

Square planar Tetrahedral

Square pyramidal Trigonal bipyramidal 6

21 Octahedral Geometry is important: Iron Proteins

Trigonal Tetragonal Octahedron Tetrahedron Bipyramide Pyramide

Rubredoxin 3,4-Protocatechoate Tyrosine Lipoxygenase Dioxygenase Hydroxylase 22 EI Solomon, et al. (2000), Chem. Rev., 100, 235–350 Color and Magnetism

Co[(NH3)6]Cl3 Co[(NH3)5Cl]Cl2

Alfred Werner (University of Zürich/CH, Nobel Prize in Inorganic Chemistry, 1913)

23 Remember: Valence Bond Theory L. Pauling

3+ 3- 6 [Co(NH3]6 and [CoF6] d

3d 4s 4p 4d

3- [CoF6] sp3d2 d d d d 2 d 2 2 xy xz yz z x -y F- F- F- F- F- F- paramagnetic, S=2

3+ [Co(NH3)6] d2sp3

NH3 NH3 NH3 NH3 NH3 NH3 diamagnetic, S=0

sp3d2 and d2sp3 hybridization 24 Color and Magnetism Variable Spin States of Metal Centers For a d5 configuration, Fe(III)

High-Spin, S= 5/2 Low-Spin, S= 1/2

Depending on the METAL ION ENVIRONMENT, balance of Crystal Field Splitting, 10Dq and Spin-Pairing Energy, P

10Dq >  10Dq < 

Strong Field Weak Field

High Spin Low-Spin 25 Metals – Biological Functions

Metal Function, Enzymes Na Charge Carrier, Osmolysis/equilibrium K Charge Carrier, Osmolysis/equilibrium Mg Structure, ATP/ThDP Binding, Photosynthesis,... Ca Structure, Signaling, Charge Carrier

V Nitrogen Fixation, Oxidases, O2 Carrier Cr Unknown! (glucose metabolism ???) Mo Nitrogen Fixation, Oxidoreductase, O-Transfer W Oxidoreductases, Acetylene Hydratase Mn Photosynthesis, Oxidases, Structure,... - Fe Oxidoreductase, O2 Transport + Activation,e -Transfer,...

Co Oxidoreductase, Vitamin B12 (Alkyl Group Transfer) Ni Hydrogenase, CO Dehydrogenase, Hydrolases, Urease - Cu Oxidoreductases, O2 Transport, e -Transfer Zn Structure, Hydrolases, Acid-Base Catalysis... 26 Oxidation States of Metals in Biology

Metal Valence state (Electron configuration) Na Na(I) K K(I) Mg Mg(II) Ca Ca(II) V V(V)=(d0), V(IV)=(d1), V(III)=(d2) Cr Cr(III)=(d3),Cr(IV)=(d2),Cr(V)=(d1) Mo Mo(III)=(d3),Mo(IV)=(d2),Mo(V)=(d1), Mo(VI)=(d0) W W(IV)=(d2) ,W(V) =(d1), W(VI)=(d0) Mn Mn(V)=(d2),Mn(IV) =(d3),Mn(III) =(d4),Mn(II) =(d5) Fe Fe(V)=(d3), Fe(IV)=(d4),Fe(III)=(d5),Fe(II)=(d6), Fe(I)?=(d7) Co Co(III)=(d6), Co(II)=(d7), Co(I)=(d8) Ni Ni(III)=(d7), Ni(II)=(d8), Ni(I)=(d9) Cu Cu(III)=(d8), Cu(II)=(d9), Cu(I)=(d10) Zn Zn(II) =(d10) 27 Exogenous ligands

Ligand pKa - 2- Acid/base H2O/OH ,O 14,~34 - 2- HCO3 /CO3 10.3 2- 3- HPO4 /PO4 12.7 - H3CCOO /H3CCOOH 4.7 - HO2 /H2O2 11.6 + NH3 /NH4 9.3 - N3 /N3H 4.8 F-, Cl- Br-, I-/XH 3.5, -7, -9, -11

Neutral O2, CO, NO, RNC d+ d- H n+ 28 M O Hd+ Modulation of pKa + n+ -H - n+ H2O + M HO -M +H+

Metal pKa none 14.0 2+ Ca 13.4 4 orders of 2+ Mn 11.1 magnitude ! Cu2+ 10.7 Zn2+ 10.0

d+ d- H n+ M O Hd+ 29 Kinetic Control

-H2O [Mn+(H O) ] [Mn+(H O) ] 2 m 2 m-1 +H2O -1 Metal k (s ) K+ 1x109 Ca2+ 3x108 Mn2+ 2x107 Fe2+ 4x106 15 orders of Co2+ 3x106 magnitude! Ni2+ 4x104 Fe3+ 2x102 3+ -6 Co <10 30 Water exchange rates M. Eigen, Nobel Prize Lecture 1967

Expressed as lifetime of complexes Useful to look at reactivity in ligand exchange reactions - catalysis

inert labile

31 Stability of Metal Ion Complexes: The Irving-Williams Series

Stability order for high-spin divalent metal ion complexes: Peak at Cu(II), Minimum at Mn(II) 32 Strong chelating ligand: EDTA

N N HOOC COOH HOOC COOH +M

-4H+

Hexadentate Ligand => strong complexing agent; can be applied to remove metal ions from biological samples (proteins, nucleic acids). 33 Protein Chelate: Bacterial Metallothionein (MT)

• 55 amino acids • One domain B • Not only Cys, but C D also 2 His A • Cluster similar to mammalian MT: Essentially a distorted piece of Zn Cys His 4 9 2 mineral (ZnS) cluster

C. A. Blindauer et al. (2001) PNAS 98, 9593-9598. 34 Biological Chelate: Siderophores

Extremely stable complex of Enterobactin/Fe3+ K~ 1049

Release of Fe through a) degradation of ligand, or b) protonation and reduction to Fe2+ which binds much weaker to the siderophore.

35 Hard and Soft Acid-Base (HSAB) Principle

„Hard“ Ligands prefer „hard“ Metal ions Ionic Bonds „Soft“ Ligands prefer „soft“ Metal ions Covalent Bonds Metal Ligand Hard Hard + + + 2+ 2+ - - 2- H , Na , K , Mg , Ca H2O, OH , R-COO , CO3 2+ 3+ 3+, 3+ - - Mn , Cr , Co Fe NH3, NO3 , R-NH2, R-O , ROR

Borderline Borderline 2+ 2+ 2+ 2+ 2+ - 2- - Fe , Ni , Zn , Mg , Ca NO2 , N2, SO3 , N3 , Ph-NH2 Co2+, Cu2+ Imidazole

Soft Soft +, 2+ + 2+ 2+ - - - 2- Cu Pt , Au , Hg , Cd R2S, RS , R3P, CN , SCN , O 2- - - S , R , H 36 Modulation/tuning of Redox Potentials E1/2

O- X X Cu Cu N N N R 2

- X=O : E1/2 = -1.21 V R=CH3 : E1/2 = -0.90 V - X=S : E1/2 = - 0.83 V R=C2H5 : E1/2 = -0.86 V R=i-Pr : E = -0.74 V Soft Ligand (RS-) 1/2 stabilizes Cu(I) state R=t-Bu : E1/2 = -0.66 V Positive Potential Steric hindrance forces tetrahedral geometry,

stabilizes Cu(I) 37 Influence of Protein Environment

• Stabilization of unfavorable metal-ligand combinations =2-5 • Low polarity  Hydrophobic chemistry • Preformed sites  „Entatic State“ • Substrate specific channels and bindungs sites • Fine-tuned acid/base chemistry • Local production of intermediates – transition states

38 Holm, R.H.; Kennepohl, P.; Solomon, E.I. (1996) Chem. Rev., 96, 2239 Metal Site 1: Blue Cu(II)/(I) Site (Plastocyanin) Function: Electron Transfer Protein/Photosynthesis

Covalent Cu-Cys -bond is mainly responsible for its unique properties EI Solomon, Inorg. Chem. 2006, 45, 8012-8025

41% Cu 38-45% S PDB Code: 1PLC HC Freeman, 1978 Cu(II) Spin-Distribution 39 Metal site 2: Heme Fe(III)/(II)

N N 199.4 pm His 200.9 pm His III II Fe e- Fe

NHis NHis

2 Reorganization energy  S(DRML) In Cytochromes  4-5 kcal/mol

Sigfridsson, E.; Olsson, M.H.M.; Ryde, U. (2001) J. Phys. Chem. B, 105, 5546 40 Metal Site 3: Iron – Sulfur centers (FeS)

Rubredoxin [3Fe-4S]

[2Fe-2S] Ferredoxin

[4Fe-4S]

[2Fe-2S] Rieske center

41 Modulation of Redox potentials (H bridges)

2Fe-2S Ferredoxin 2Fe-2S Rieske

E0‘ ~ -400 mV E0‘ ~ +280 mV

(+150 mV without H bridges)

42 (a) Stephens, P.J.; Jollie, D.R.; Warshel, A. (1996) Chem. Rev., 96, 2491 (b) Link, T.A. (1999) Adv. Inorg. Chem., 47, 83 Proteins Tune the Properties of Metal Ions

Coordination number – The lower the higher the Lewis acidity Coordination geometry – Proteins can dictate distortion – Distortion can change reactivity of metal ion Weak interactions - Second Shell Effects – Hydrogen bonds to bound ligands – Hydrophobic residues: dielectric constant can change stability of metal-ligand bonds

43 Conclusion

The structural and functional properties of metal ions in biological systems can be understood by combining the principles of coordination chemistry with the knowledge of the unique environment created by biomolecules

Bo G. Malmström, Göteborg, 1927-2000 44 The Chelate Effect (Entropy)

Multidentate vs unidentate ligands (number of binding sites)

2+ 2+ (1) [Cu(H2O)6] + en  [Cu(H2O)4(en)] + 2H2O

0 0 0  DG   DH   DS  0 0 0 2 K  exp   exp exp  DG  DH TDS CuH O enH O  RT   RT   R  log K  log 2 4 2 10.6       CuH2O6 en DH 0  54kJ mol-1 DS 0  23Jmol-1 K 1

2+ 2+ (2) [Cu(H2O)6] + 2NH3  [Cu(H2O)4(NH3)2] + 2H2O

2 Cu H O NH H O 0 -1 0 -1 1   2 4  3 2  2  DH  46kJ mol DS  8.4Jmol K log K  log 2  7.7 CuH2O6 NH3 

45 Multidentate Ligands

Monodentate: F-, Br-, Cl-, I-, O2-, S2-, R-O-, Py N

Bidentate : Ethylendiamine, MNT, Glycine

NC CN COO-

H N H2N CH2 2 NH2 -S S-

N Tridentate : Triethylentriamine (TREN) H NH2 NH2

Tetradentate : Porphyrin NH HN