Lecture 9

Redox metallo-biochemistry

(continued)

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

Absorption spectra of oxidized (Fe(III) and reduced (Fe(II)) horse cytochrome c.

• classified by 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

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)

•Conserved from bacteria to Man

horse heart cytochrome cBushnell, G.W.,  Louie, G.V.,  Brayer, G.D. J.Mol.Biol. v214 pp.585-595 , 1990

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 CN5, axial ligand is a CysCys

• 66thth site for site for substrate/oxygen substrate/oxygen bindingbinding

• Hydroxylates Hydroxylates camphorcamphor

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.

Fe2S2(Cys-S)4

Fe2S2(Cys-S)2-(His-N)2: Rieske proteins

Fe4S4(Cys-S)4

Fe3S4(Cys-S)4

1 awd: CHLORELLA FUSCA

1fda: Azotobacter vinelandii

1rfs: Spinach

Fe-S clusters can be easily synthesised from Fe(III), sulfide and organic thiols, but are prone

to rapid oxidation

Richard Holm Self-assembly of Fe-S clusters

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:

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

P cluster of nitrogenase

FeMoCo cofactor cluster of nitrogenase

Nitrogenase (Klebsiella pneumoniae)

• Catalyses nitrogen fixation

•N2 + 8H+ + 8e- + 16 ATP → 2NH3 + H2 + 16ADP + 16 Pi

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

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

2.9 Å

2.11 Å

Amicyanin (pdb 1aac) from Paracoccus denitrificans

Blue copper proteins: coordination geometry

Angles also deviate strongly from ideal tetrahedron(84-136°)

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