Download [Ru(NH 3 ) 5 (His33)] 2+ @ 18 Å from heme

Electron Transfer Through Proteins: Harry Gray and Jay Winkler, Cal Tech
“Aerobic respiration and photosynthesis work in concert: the oxygen that is evolved by PS
organisms is the oxidant that sustains life in aerobic microbes and animals and in turn, the
end products of aerobic respiratory metabolism ( CO2 and H2O) nourish photosynthetic
organisms.”
Important terms:
kET, electron transfer rate
l, reorganization energies,
includes both inner sphere (bonds, angles)
and outer sphere (solvent, dielectric, protein)
DGo, free energy change between donor (reductant) and acceptor (oxidant) at infinite
separation. Is the driving force.
HAB, the electronic coupling matrix element, determine the frequency of crossing the
transition state from (D + A) to (D+ + A-)
A balance between nuclear reorganization and reaction driving force determines both the
transition state configuration and the height of the barrier for the ET process.
For the optimum driving force,
DGo = l ,
the reaction is activationless.
rgt
prdt
rgt
prdt
rgt
Adiabatic system:
where rgt and prdt
prdt stay on lower
electronic surface
A very bad diagram!!
Normal
free energy
region
Inverted
region
A balance between nuclear reorganization and reaction driving force determines both
the transition state configuration and the height of the barrier for the ET process.
For the optimum driving force, DGo = l ,
the reaction is activationless.
Example of the role of reorganization energy in ET rate:
Fe(3+)aq + e-  Fe(2+)aq +0.77V = Eo
Activation Energy = 0.66 eV
This corresponds to a 2.7 eV reorganization energy.
1.5 eV is change in Fe—O bond length (the inner sphere piece)
(increases 0.14 Å after reduction) and
1.2 eV from solvent repolarization (the outer sphere piece).
kET, ET rate = 100 milllisec if two species in contact.
Separated by 20 Å in a vacuum: kET, = 1017 years
Separated by 20 Å in water: kET, = 104 years
Separated by 20 Å, and tunneling through hydrocarbon:
kET, ~ msec - msec
Hence, fast biological ET must be protein mediated.
Studies of ET through proteins:
1. Attach Ru complexes to His residues on surface
(“labeled redox active molecules”)
2. Photoexcite Ru with laser
3. Detect ET event to other metal center:
measure rate of ET to protein metal site.
[Ru(NH3)5(His33)]2+ @ 18 Å from heme;
16.8 Å
Cytochrome c,
oxidized, as
Fe3+
driving force = +0.20 eV,
ET rate = 30 sec -1
To compute reorganization energy, vary DGo of reaction (how?)
From measurements on [RuL(NH3)4(His33)]2+ with Zn – cyt c
(L = pyridine, ammonia, isonicotinamide)
Result: hydrophilic Ru-ammine causes ~2/3 of reorganization energy
[Ru(NH3)5(His33)]2+ @ 18 Å from heme;
driving force = +0.20 eV for Ru(2+) Fe(3+)
ET rate = 30 sec -1
l = 1.15 eV
16.8 Å
[Ru(bpy)2(imid)(His3)]2+@ 18 Å from heme;
l = 0.74 eV
Reorganization energy for [Ru(bpy)2(imid)(His3)]2+ is
about 2/3 x less reorganization energy for
[Ru(NH3)5(His33)]2+ due to organization energy
required by water on hydrophilic ammines vs
hydrophobic bpy ligands.
General rule:
A hydrophilic site will lead to larger reorganization
energies than a hydrophobic site.
Therefore, ET reactive sites are very sensitive to
active-site environment.
But this isn’t really news, is it?
Cu(Cys)(Met)(His)2 site in azurin
[Ru(bpy)2(imid)(His)
2+
3] labeled azurin
Electron transfer with Cu(2+)(H2O)6 is very
sluggish in water because of large
structural change on reduction to Cu(+)
(what does it want to be (….?).
Hence, azurin creates a rigid Cu site that
does not change on reduction, eliminating
large reorganization energies.
And, Cu strongly coupled through Cys by
pi-bonding: effectively directs e- flow.
The fact that
the reorganization energy for electron exchange
in Cu(phen)2 2+/+
is 2 eV greater than for azurin
illustrates the important role played by
the folded protein in lowering ET barriers.
Studies of ET through proteins map the ET path:
1. Attach Ru complexes to His residues on surface (“labeled redox active molecules”).
Photoexcite Ru, “watch” ET event, measure rate of ET to protein metal site.
2. When donors and acceptors are separated by > 10 Å, the electronic coupling term (HAB)
is nearly zero due to vanishingly small overlap of their orbitals. Now the protein must
mediate the ET event.
3. This is a modification of a “superexchange” model, where D and A are coupled through a
bridging molecule and e is coupling decay parameter quantifying the effect of bridge.
4. The “superexchange” idea modified for a protein assumes that electronic coupling by
tunneling is approximated as:
HAB ~ ecovalent * e H bonds * e space
interactions
5. Now one compares the ET calculated from a path through the protein, obtained from a
search algorithm that charts a path of bonds and space in a protein, to observed ET
rate.
6. From this, a proposed ET path is identified.
7. CONCLUSION: a-helices are slower paths than b-sheets
A tunneling “timetable” plot,
Illustrates how the tunneling
time, equals 1/ kET , varies with
distance between D and A.
This calculated plot is for ET in
Ru-modified myoglobin and
cytochrome b.
The lines show predicted ET
tunneling times for
a-helices
vs b-sheet pathways.
CONCLUSION:
a-helices are slower paths
than b-sheets
kETthrough
b sheets
kETthrough
a helices
Note b-barrel:
Efficient ET mediator
Rates of reduction of Os(III), Ru(III), and Re(I) by Cu(I) in
His83-modified Pseudomonas aeruginosa azurins (Az)
(M-Cu distance approximately 17 Å) have been measured
in single crystals, where protein conformation and
surface solvation are precisely defined by high-resolution
X-ray structure determinations. The rates for electron
tunneling in crystals are roughly the same as those
measured in solution, indicating very similar protein
structures in the two states. High-resolution structures of
the oxidized (1.5 Å) and reduced (1.4 Å) states of
Ru(II)(tpy)(phen)(His83)-Az establish that very small
changes in copper coordination accompany reduction.
Although Ru(bpy)2(im)(His83)-Az is less solvated in the
crystal, the reorganization energy for Cu(I) --> Ru(III)
electron transfer falls in the same range determined
experimentally for the reaction in solution.
Cu
Therefore this work suggests that outer-sphere protein
reorganization is the dominant activation component
required for electron tunneling.
Ru2+(tpy)(phen)(His83)