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

2. Cytochromes Fe-S proteins Blue copper proteins
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: nm
b: nm
c: 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. v pp , 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,

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. Azotobacter vinelandii: 2 clusters
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 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 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