Redox metallo-biochemistry (continued) Lecture 9 Redox metallo-biochemistry (continued)

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

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)

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

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.

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. v214 pp.585-595 , 1990 Conserved from bacteria to Man

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.

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

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

Iron-sulfur proteins

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)

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

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

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

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

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

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

FeMoCo cofactor cluster of nitrogenase P cluster of nitrogenase

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

Redox potentials

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

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

Copper proteins

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

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

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

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

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

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