1.
2.
3.
4.
5.
6.
7.
Heme group vs
Tunable
No control of substrate approach
Single heme group
Solvent effect – water
No control of porphyrin ring flex
Relatively easy to fix to electrode
Fast e.t.
Proteins
tunable
Control of substrate approach
Native – intra/inter heme group e.t.
solvent – protein
control of porphyrin ring flex
?
?
Cytochrome C
Microperoxidase
protolytic
digestion of
cytochrome c
CH3
CH3
Highest rate in
Literature (2000)
was
12 to 20 1/s
CH3
H3C
400-2000 1/s
CH3
O
H3C
NH
The protein “clothing” must play a very
Important role in the action of the heme
Group!!
N
OH
4000 1/s
O
N
HN
O
CH3
CH2
OH
Heme a
Some possible differences between the heme site and the full
protein:
1. Use of the protein to channel or tunnel the charge as opposed to
through space hopping
2. Control of the shape of the porphyrin ring to facilitate concerted e-H2O
reaction
3. Control of the presentation of the active site toward the electrode and or
reactive surface
(Gray and Ellis review chapter)
E.t. often occurs between prosthetic groups separated by distances > 10
Need to consider special circumstances for this e.t.
k et  Ae

 rnn  ro 

H AB   
k et 
   RT 
2
We just saw a simplified
Hopping model
  G 


o 2
1/ 2
e
4  RT
The Marcus model
HAB describes electronic coupling between reactants & products at the transition state.

o

d

d
o
2
H AB  H AB e


Rate of decay with distance
Related to the protein medium
Close contact position
HAB magnitude depends
1.
upon Donor (reductant)- A (oxidant) separation
2.
orientation
3.
nature of intervening medium
Harry Gray et al, Inorganic Chemistry 43(12) 3593: Electron-transfer Chemistry of Ru-Linker-(Heme)Modified Myoglobin: Rapid Intraprotein Reduction of a photogenerated Porphyrin cation radical
Porphyrin = acceptor
Ru* metal complex (donor)
Myoglobin surroundings
Monitor E.T. by loss of the peaks at 250 or 400
Calculate rate
From decay
Gray Review Article
 d  d o  

 H AB o e  2 
2


1/ 2
  G o 


   

4  RT
k et 
e



 RT 
2
  d  d o  

ln k et  ln stuff   
2



o

d

d
o
2
H AB  H AB e


H AB   
k et 
   RT 
2
  G 


o 2
1/ 2
e
4  RT
(Gray and Ellis review chapter)
k et  Ae

 rnn  ro 

H AB   
k et 
   RT 
2
1/ 2

o

d

d
o
2
H AB  H AB e

e


HAB magnitude depends
1.
upon Donor (reductant)- A (oxidant) separation
2.
orientation
3.
nature of intervening medium

  G o
4  RT

2
J. AM. CHEM. SOC. 2007, 129, 3906-3917
Brian Hoffman et al, JACS 2007,
Cyt b and hb inter (between) vs intra (within) et
ET depends on docking – must
have propionates in close
proximity
Active propionates close
Myo cyt c complex
inactive
(Gray and Ellis review chapter)
k et  Ae

 rnn  ro 

H AB   
k et 
   RT 
2
1/ 2

o

d

d
o
2
H AB  H AB e

e


HAB magnitude depends
1.
upon Donor (reductant)- A (oxidant) separation
2.
orientation
3.
nature of intervening medium
1. as a charge conducting media

  G o
4  RT

2
Gray and Ellis Review Article
E.T. pathway can be considered
to be a covalent bond, H bond
or through space jump
Each has own decay  factor.
Dominant tunneling pathways in
proteins are largely composed
of bonded groups
Less favorable through-space
interactions become important
only when a through-bond
pathway is too long.
Gray Ellis Review article
From the variety of modified myoglobin articles:
For myoglobin the reaction requires the loss of water from the iron site,
But this loss is not rate determining
Cytochrome c has also been Ru modified and
modeled with pathways
– covalent bond, H-bond, through-space jump
for e.t. from a histidine to the heme site
Reasonable agreement results
Are obtained
Gray and Ellis review article
(Gray and Ellis review chapter)
k et  Ae

 rnn  ro 

H AB   
k et 
   RT 
2
1/ 2

o

d

d
o
2
H AB  H AB e

e


  G o

Other researchers have suggested that the electron transfer rate is
Affected by associated chemical reactions
EC – mechanism we have already studied
CE – new mechanism
4  RT

2
Some possible differences between the heme site and the full
protein:
1. Use of the protein to channel or tunnel the charge as opposed to through
space hopping
2. Control of the shape of the porphyrin ring to facilitate concerted e-H2O
reaction
3. Control of the presentation of the active site toward the electrode and or
reactive surface
For Proteins E.T. can be “gated” by a CE mechanism involving conformational
change
-8.00E-01
-6.00E-01
-4.00E-01
-2.00E-01
I
0.00E+00
2.00E-01
4.00E-01
6.00E-01
8.00E-01
1.00E+00
-0.8
-0.7
-0.6
-0.5
V
-0.4
1 cm2; 1 M/L, A=B (K=10-4; kf varies from 100 to 108)
B+e = C (ket=104); Alpha = 0.5; 1V/s, Eo= -0.2
-0.3
-0.2
-0.1
0
Tuning the Rate and pH Accessibility of a Conformational Electron Transfer Gate; Saritha Baddam and
Bruce E. Bowler*, Inorganic Chemistry 2006, 45, 6338
E
C
C
E
CE
Dominate pathway implies “conformational gating”
Cytochrome c system
For Proteins E.T. can be “gated” by a CE mechanism involving conformational
change
This suggests that the rte of e.t. can be controlled by controlling the
conformational change
1. change diameter of cavity the protein has to move in (sol gel encapsulation)
2. Change viscosity
Sol gel modified electrodes:
=utter simplicity
Si OH  4,aq  SiOx  OH  4 x  xH 
x
Sol-gel Science, The Pjysics and Chemistry of Sol-Gel
Processing, C. Jeffrey Brinker, George W. Scherer
Hoffman et al Proceedings of the National Academy of Science 2005
et
et
1. forward and reverse ET response to changes in
viscosity is different for each system.
2. For the most rigid protein complex (Hb hybrid) kb is
essentially invariant with viscosity, while kf falls
significantly.
3. For the [ZnCcP,Fe3Cc] complex, with small
increases in viscosity, kf is invariant with increasing
viscosity; kb is nearly so
4. At higher viscosities, kb falls more rapidly than
kf, in contrast to the hybrids but similar to the
complexes with Fe3b5.
5. Among the issues to be addressed are the degree
to which the addition of glycerol may be
1. (i) changing the energy landscapes
themselves, not merely the rates with which
the landscapes are traversed and
2. (ii) influencing water activity.
Hemoglobin-like structure
e
 H 2 O,
Behaves as
A concerted
process

 
Fe 3 (t 25g )  H 2 O  e  Fe 2  t 26g  H2 O
Low spin
High spin
Wittenberg, PNAS, 1970, 67,4 , 1846
Oxy Heme
Met Heme
Deoxy Heme
Alteration results in change in electron configuration and bond length changes with
proximal histidine H102
Oldham JACS 2003, 125, 16387
Note the flexing of the porphyrin
Ring on various types of binding
To the central iron. This flexing
Results in different reactivities
of the iron site by
a) changing the formal potential
associated with the iron
b) changing the binding of water
to the iron site
Which change affects the rate of electron transfer the most?
The shape of the porphyrin ring flex can be fixed by cross linking protein subunits
Look for change in potential and see if it correlates with resulting change in rate –
if so suggests outer sphere electron transfer (concerted eH)
if not suggests two separate steps E, H, with the latter rate controlling as an
inner sphere mechanism
Oxy (Fe(II)(t62g)
deoxy (Fe(II)
K
 
R (flattened)

For an animation see
http://en.wikipedia.org/wiki/hemoglobin
Something like this shape is adopted
By met (Fe(III)
T (domed)
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hemoglobin/MetalComplexinBlood.html
Eo=0.05V
alpha1
Intra (within) e.t.
inter
inter
6/hr
collisional
Eo=0.11V
beta1
et
Kiger, Laurent and MichaelC. Marder, Electron Transfer Kinetics
between Hemoglobin Subunits, Journal of biological Chemistry,
276, 51,2001, 47937
In hemoglobin, 4 heme sites:
1
1
Same side
2
2
Only the  1
2
e.t. is probable (20 heme
edge to heme edge) since the
remainder are too distant.
Some of the other
conclusions drawn by
the substituted metal
work is that the multiple
e.t. (4e) is driven by
collision and requires
flexibility of the central
pocket
Hemoglobin
1. Determine Shift in potential with alteration in heme site structure
2. Determine if k11 is dependent on k
If so is an “outer sphere” reaction and eH remains concerted
log k12 
1
log k11  log k 22  16.9  E o 

2
3. Determine if the apparent binding constant of water has
changed
Work of Simona Dragan
Spectroelectrochemistry
UV-Visible Spectroscopy and Controlled Potential Coulometry
Optically Transparent Thin Layer Electrochemical Cell
V = 50.1 + 0.5 mL
Counter
electrode:
Pt wire
Counter
electrode:
Pt wire
Quartz
Working
electrode:
Pt minigrid
OTTLE
characteristics
Solution out
Reference
electrode:
Ag/AgCl
Cell
compartment
Solution in
Teflon spacer
OTTLE a la Heineman
l = 0.5 mm
1. Renewable
solution
2. Restricted
volume
3. Rapid
electrolysis
4. Resistance is
low
5. Rigorously
oxygen free
Met (Fe(III),R
V = 50.1 + 0.5 mL
Counter
electrode:
Pt wire
4 H2 O
l = 0.5 mm
Working
electrode:
Pt minigrid
Ru NH3  6
3
Ru NH3  6
2
Counter
electrode:
Pt wire
Teflon spacer
Deoxy, (Fe(II), T
Hemoglobin
1. Determine Shift in potential with alteration in heme site structure
2. Determine if k12 is modulated by by delta E
log k12 
1
log k11  log k 22  16.9  E o 

2
3. Determine if the apparent binding constant of water has changed
Change stability (delta E) by altering salt bonds which affect the quaternary transition
Equilibrium Redox Measurements
XLHbA 0.1 mM, Ru(NH3)6Cl3 0.5 mM/KCl 0.05 M, MOPS 0.05 M, pH 7.1
0.7
MetHb
0.7
DeoxyHb
0.6
0.6
0.05 V
-0.05 V
-0.06 V
-0.07 V
-0.08 V
-0.10 V
-0.20 V
0.4
0.3
0.5
Absorbance
Absorbance
0.5
0.4
0.3
0.2
0.2
0.1
0.1
0.0
0.0
350
Soret bands
400
450 500
, nm
550
430 nm
600
406 nm
-0.4 -0.3 -0.2 -0.1 0
E, V vs . Ag/AgCl
0.1
The preceding slide should remind you of:
1
1  exp
 nF 

Eo
 E
 RT  electrode

Ox




Ox 
electrode
initial
1.2
[Remaining Ox]/[Initial Ox]
1
0.8
0.6
0.4
0.2
0
-0.25
-0.2
-0.15
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
E-Eo (V)
An electrochemical “titration curve” centered on Eo
Time-based Measurements
XLHbA 0.1 mM, Ru(NH3)6Cl3 0.5 mM/KCl 0.05 M, MOPS 0.05 M, pH 7.1
Absorbance
0.8
406 nm
430 nm
0.6
Oxidation
Reduction
E = 0.3 V vs. Ag/AgCl
E = -0.5 V
406 nm
0.4
0.2
430 nm
0.0
0
5
2.5
25
30
first rate order kinetics
3.0
430 nm
430
nm
2.0
20
time, min
autocatalytic mechanism
3.0
406 nm
2.5
406
406nm
nm
ln(c0/c)
103xrate/c0, s-1
15
10
1.5
1.0
2.0
430 nm
1.5
1.0
0.5
0.5
0.0
0
150
300
time, s
450
k = 0. 0474 s-1
0.0
0
20
40
time, s
60
Ru NH3  6  HemeFe II
3
Ru NH3  6
3
k
12


Ru NH3  6  HemeFe III
2
o
2




e 
Ru NH3  6
k ,E
k11 =1.01 M-1s-1
k11 =0.69 M-1s-1
k11 =0.08 M-1s-1
Literature(0.144)
log k12 
1
log k11  log k 22  16.9  E o 

2
Value obtained for Native Heme is consistent with other experiments
Type of Experiment
k11 (1/Ms)
Hemoglobin
Thin layer Spectrophotometric
1.01
1.37x10-10
Pt
Brilliant Cresol Blue
3.9x10-5
Pt
Methylene Green
Gold
Alkanethiol
0.144
Pt
Didodecyldimethyl-ammonium bromide
0.002 to 0.121
Alpha-H
0.08
Poor fit of Beta to the Marcus cross reaction expression implies that the
concerted eH2O reaction is not longer operative.
Test this presumption by determining the water dissociation constant
Beta-H
0.69
T
II
Fe III  H2 OT  e 
Fe
T  H2 O

K
measure
nmax  1 
A measure of the binding of water
 EoF 

   E o F 



 RT 


RT

e
K RH2O  e  

3 1 
 1
2


K T , H2 O
 K T , H2 O


 



 EoF 

   E o F 



 RT 


RT



e
K RH2O  e
1

 1
2

K T , H2 O   K T , H2 O


 



Varied
1 unknown
XL-HbA
HbA
XL-HbA
2
log([O]/[R])
Nernst Equation
MOPS 0.05 M, pH 7.1
3
1
E  Eo 
0
0.058   R 
log

  O 
n
E  Eo 
-1
E0
n
n
  O 
E
E o  log

  R 
0.058
0.058
-2
-3
-0.1
2.0
0.0
0.1
E, V vs. NHE
0.2
0.3
Slope related
To total number
Of electrons
transferred
n5
n50 and nmax
0
1.5
1.0
Plot nmax as a function of the
Formal potential
0.5
0.0
-0.1
0.058   O 
log

  R 
n
0.0
0.1
E, V vs. NHE
0.2
0.3
Best fit a curve to get KT
Bonaventura
Our data

 
XL, domed
XL, flat
K R , Fe( III )  34; lessaffinity for water
K R  20(12    20)
Same for native
Suggests rate for βXL is controlled by access of
Water not by electronic factors in the heme site