1 / 29

Lecture 9

Lecture 9. Redox metallo-biochemistry (continued). e - transfer proteins. Cytochromes Fe-S proteins Blue copper proteins. Kinetics of electron transfer reactions.

mircea
Download Presentation

Lecture 9

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Lecture 9 Redox metallo-biochemistry (continued)

  2. e- transfer proteins Cytochromes Fe-S proteins Blue copper proteins

  3. Kinetics of electron transfer reactions • Electron transfer between 2 metal centers can be either inner-sphere (via a bridging ligand) or outer-sphere (no bridging ligand, coordination spheres remain the same for both metal ions) • Only outer-sphere known for metalloproteins • Reasonably fast (> 10 s-1) over large distances (up to 30 Å) • Can be rationalised by Marcus Theory • Qualitatively: e- transfer is fast if the states before and after the redox reactions are similar (reorganisation energy is small)

  4. Cytochromes • Name comes from the fact that they are coloured • Differ by axial ligands and whether covalently bound • Involved in electron transfer (a,b,c) or oxygen activation (P450) • Essential for many redox reactions

  5. UV-Vis Spectra of cytochromes • classified by a bands: • a: 580-590 nm • b: 550-560 nm • c: 548-552 nm • (there’s also d and f) • all involved in electron transfer, all CN6 • P450: 450 nm: • Oxygen activation; CN5 Absorption spectra of oxidized (Fe(III) and reduced (Fe(II)) horse cytochrome c.

  6. Cytochrome c • Small soluble proteins (ca. 12 kDa) • Near inner membrane of mitochondria • Transfers electrons between 2 membrane proteins ( for respiration) • Heme is covalently linked to protein via vinyl groups (thioether bonds with Cys) • 1 Met and 1 His ligand (axial) horse heart cytochrome c Bushnell, G.W.,  Louie, G.V.,  Brayer, G.D. J.Mol.Biol.v214pp.585-595 , 1990 • Conserved from bacteria to Man

  7. Cytochromes b • Heme has no covalent link to protein • Two axial His ligands • Shown is only soluble domain; the intact protein is bound to membrane F Arnesano, L Banci, I Bertini, IC Felli:The solution structure of oxidized rat microsomal cytochrome b5. Biochemistry (1998) 37, 173-84.

  8. Not for electron transfer:the cytochromes P450 • CN5, axial ligand is a Cys • 6th site for substrate/oxygen binding • Hydroxylates camphor P450Cam

  9. Tuning of heme function • In (deoxy)hemoglobin, Fe(II) is 5-coordinate • Must avoid oxidation to Fe(III) (Met-hemoglobin) • Neutral His ligand: His-Fe(II)-porphyrin is uncharged: Favourable • P450: Catalyses hydroxylation of hydrophobic substrates. Also 5-coordinate • 1 axial Cys thiolate ligand (negatively charged): Resting state is Fe(III), also uncharged • In cytochromes, CN=6: No binding of additional ligand, but very effective 1 e- transfer

  10. Iron-sulfur proteins

  11. Fe-S proteins • Probably amongst the first enzymes • Generally, Fe, Cys thiolate and sulfide • Main function: fast e- transfer • At least 13 Fe-S clusters in mitochondrial respiration chain • Rubredoxins: mononuclear FeCys4 site • Ferredoxins: 2,3 or 4 irons • Other functions: Aconitase: An isomerase IRE-BP: An iron sensor (see lecture 5)

  12. Rubredoxins: FeCys4 X-ray Structure of RUBREDOXIN from Desulfovibrio gigas at 1.4 A resolution. FREY, M., SIEKER, L.C., PAYAN, F.

  13. 1rfs: Spinach Fe2S2(Cys-S)4 1 awd: CHLORELLA FUSCA Fe2S2(Cys-S)2-(His-N)2: Rieske proteins Fe3S4(Cys-S)4 Fe4S4(Cys-S)4 1fda: Azotobacter vinelandii

  14. Fe-S clusters can be easily synthesised from Fe(III), sulfide and organic thiols, but are prone to rapid oxidation Self-assembly of Fe-S clusters Richard Holm

  15. Delocalisation of electrons: Mixed valence • localized Fe3+ (red) and localized Fe2+ (blue) sites, and • delocalized Fe2.5+Fe2.5+pairs (green) • Why e- transfer is fast: • Clusters can delocalize the “added” electron • minimizes bond length changes • decreases reorganization energy

  16. Fe-S proteins often contain more than one cluster: Azotobacter vinelandii: 2 clusters

  17. The five Fe-S clusters of the Fe-only hydrogenase from Clostridium pasteurianum • Activation of H2 • Active site (binuclear Fe cluster) on top • The other five Fe-S clusters provide long-range electron transfer pathways Pdb 1feh

  18. FeMoCo cofactor cluster of nitrogenase P cluster of nitrogenase

  19. Nitrogenase (Klebsiella pneumoniae) • Catalyses nitrogen fixation • N2 + 8H+ + 8e- + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi

  20. Redox potentials

  21. Tuning of redox potentials • For both heme proteins and Fe-S clusters, ligands coarsely tune redox potential • In [4Fe-4S] clusters, proteins can stabilise a particular redox couple • Further effects (a) solvent exposure of the cluster (b) specific hydrogen bonding networks especially NH-S bonds (c) the proximity and orientation of protein backbone and side chain dipoles (d) the proximity of charged residues to the cluster

  22. Tuning of redox potentials • Bacterial ferredoxins and HiPIPs: Both have Fe4S4Cys4 clusters • -400 mV vs. +350 mV • Ferredoxins: [Fe4S4Cys4]3-→ [Fe4S4Cys4]2- • HiPIPs: [Fe4S4Cys4]2- → [Fe4S4Cys4]1- • HiPIPs are more hydrophobic: Favours -1 • NH...S bonds: 8-9 in Fd, only 5 in HiPIPs • Compensate charge on cluster; -3 favoured *) HiPIP: high potential iron-sulfur proteins

  23. Copper proteins

  24. Copper proteins • Oxidases • Cytochrome oxidase(s) • Enzymes dealing with oxides of nitrogen • Blue copper proteins • Superoxide dismutase • Tyrosinase • Caeruloplasmin

  25. Principles • Cu(II) forms the strongest M(II) complexes (see Irving Williams series) • Cu(I) also forms stable complexes • The Cu(I)/Cu(II) redox couple: 0.2V-0.8V • Most Cu proteins either extracellular or membrane-bound • Many Cu proteins involved in electron transfer

  26. Preferred geometries • Cu(II): Tetrahedron • Cu(I): trigonal planar or 2-coordinate

  27. Blue copper proteins • Azurin, stellacyanin, plastocyanin • Unusual coordination geometry: Another example for how proteins tune metal properties • Consequences: • Curious absorption and EPR spectra • High redox potential (Cu(I) favoured) • No geometric rearrangement for redox reaction: Very fast

  28. Blue copper proteins: coordination geometry 2.11 Å 2.9 Å Angles also deviate strongly from ideal tetrahedron (84-136°) Amicyanin (pdb 1aac) from Paracoccus denitrificans

  29. Key points • Properties such as redox potentials are tuned by proteins • Coarse tuning by metal ligands • Charge imposed by ligand can favour particular oxidation state • Geometry can be imposed by protein: Can favour particular oxidation state, and also increase reaction rate • Fine tuning by “second shell”: hydrophobicity, hydrogen bonds, charges in vicinity

More Related