Transcript [Fe 4 S 4 Cys 4 ] 1

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
• 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
horse heart cytochrome c
Bushnell, G.W., Louie, G.V., Brayer,
with Cys)
G.D. J.Mol.Biol. v214 pp.585-595 ,
1990
• 1 Met and 1 His ligand
•Conserved from bacteria to (axial)
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
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
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 longrange 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
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