Cv_reac_5c2.ppt / 040608 * : S=1/2 active Ni a -S 1931 Ni a -C* 1950 Ni a -SR 1936 Activation/...
-
date post
22-Dec-2015 -
Category
Documents
-
view
216 -
download
0
Transcript of Cv_reac_5c2.ppt / 040608 * : S=1/2 active Ni a -S 1931 Ni a -C* 1950 Ni a -SR 1936 Activation/...
cv_reac_5c2.ppt / 040608
* : S=1/2
activeNia-S1931
Nia-C*1950
Nia-SR1936
Activation/Inactivation
H2
H2
inactive
Niu*1945
Niu-S1948
Nir*1943
Nir-S1931
secs
What leads to oxidative inactivation, and how is it reversed ?
‘Unready’Ni-A
‘Ready’Ni-B
?
Ni(III)
Ni(II)
v. slow
Hydrogenase adsorbed on PGE electrode, pH 9.0,
H2 at approx 1 atm,
Scan rate 0.3 mV/s. electrode rotating at 1500
rpm, temp 45 oC.
Jones et al. JACS 125, 8505 (2003)
-0.6 -0.4 -0.2 0 0.2 0.4
Potential / V vs SHE
H2 catalytic oxidationcurrent
InactiveActive
NiII Fe NiIII FeOH
Oxidative cycles at slow scan rates reveal the anaerobic interconversion between active and the ‘Ready’ state of NiFe Hydrogenase.
Anne Jones
-0.6 -0.4 -0.2 0 0.2 0.4
Potential / V vs SHE
H2 oxidationactivity
Inactivate
Activate
0 500 1000 1500 2000 2500-0.6
-0.4
-0.2
0.0
0.2
500 1000 1500 2000 2500-1
0
1
2
3
4
i(A
)P
oten
tial /
V vs.
SH
E
Time / s
log
( i li m
-i t)
-0.0008 V 0.042 V 0.092 V 0.142 V
(C)
(B)
(A)
0 500 1000 1500 2000 2500-0.6
-0.4
-0.2
0.0
0.2
500 1000 1500 2000 2500-1
0
1
2
3
4
i(A
)P
oten
tial /
V vs.
SH
E
Time / s
log
( i li m
-i t)
-0.0008 V 0.042 V 0.092 V 0.142 V
(C)
(B)
(A)
time/s
Study kinetics ofinactivationby applying steps tohigh -potential
Rates of oxidativeinactivation are independent of potential (electrodicdriving force)
(results at pH 8.8)
Jones et alJACS, 2003
potential steps
T i m e ( s )
( B )
1
3
2
4
0 5 0 1 0 0 1 5 0
- 6
- 7
- 8
0 5 0 1 0 0 1 5 00 . 0 0
0 . 2 5
0 . 5 0
( A )
1
2
34
- 0 . 4P o t e n t i a l ( V )
0
1
34
2
T i m e ( s )
( B )
1
3
2
4
0 5 0 1 0 0 1 5 0
- 6
- 7
- 8
0 5 0 1 0 0 1 5 00 . 0 0
0 . 2 5
0 . 5 0
( A )
1
2
34
- 0 . 4P o t e n t i a l ( V )
0
1
34
2
0 5 0 1 0 0 1 5 00 . 0 0
0 . 2 5
0 . 5 0
( A )
1
2
34
- 0 . 4P o t e n t i a l ( V )
0
1
34
2
Study kinetics of activationby applying stepsto low -potential
Enzyme is easily reactivated
Rate increasesas potential is lowered (i.e. as driving force is raised)
Jones et al JACS, 2003
semi-log plots
increasingdriving force(more negativepotentials)
Inactive
Active
Decreasing oxidation level
Niu* Nir*
Niu-S Nir-S Nia-S
Nia-C*
Nia-SR
Inactive
Active
Decreasing oxidation level
Niu* Nir*
Niu-S Nir-S Nia-S
Nia-C*
Nia-SR
‘unready’ ‘ready’
Ni(III)
Ni(II)
E
C
NiIII –OH Fe
NiII –OH Fe NiII(H2O) Fe
e-
H+
ADDING O2 at -0.158 V vs. SHE, pH 9, 45 oC. 0.3 mV s-
1.
O2 attacks site directly,and enzyme inactivated much faster than observed for anaerobic reaction. But most activity is regained rapidly when the scan direction is reversed, at the same potential as for the anaerobic inactivation.
inject O2
i (A
)
Potential /V vs SHE
-0.6 -0.4 -0.2 0.40.20.0
1.6
1.2
0.8
0.4
0.0
Lamle et al JACS 2004
0 500 1000 1500 20000
10
20
30
40
curr
ent (
A)
Time (s)
Fast phase of reactivation (READY)
Slow phase of reactivation (UNREADY)
-88 mVstep to -88 mV to reactivate
step to 242 mV add O2 then purge headgas with H2
(iii)
The O2 injection and potential-step sequence experiment; pH 6, 45 oCO2 added to enzyme whileit turns over H2
O2 was injected at 42 mV / H2
O2 was injected at 42 mV under N2
O2 was injected at 242 mV under N2
Nor
mal
ized
i
0.50
1.00
0.75
0.25
0.00
Time (s)
200015001000500
O2 was injected at 217 mV under H2
Injection O2 under Ar or N2 generates more ‘Unready’ than injecting O2 under H2 and there is markeddependence on electrode potential
-4.8
-5.2
-6.0
-5.6
3.1 3.2 3.3 3.4
1 /T (K-1)
log
( k/
T)
-5.4
-5.8
-6.2
-5.0
-4.8
-5.2
-6.0
-5.6
3.1 3.2 3.3 3.4
1 /T (K-1)
log
( k/
T)
-5.4
-5.8
-6.2
-5.0
Activationplot forslow phase
H 88 kJ/mol
t1/2 = 280 secat 45 oC
Lamle et al JACS 2004
E vs SHE
% U
nre
ady
Under N2
Under H2
0
20
40
60
80
100
-0.05 0.0 0.05 0.1 0.15 0.2 0.25 0.3 0.35-0.1
Unready is formed when the active enzyme reacts with O2 and there are not many electrons available !
+ CO before O2
pH 9
Lamle et al JACS 2004
Hypothesis...Hypothesis...When active enzyme is When active enzyme is drained of electrons, drained of electrons,
exposure to Oexposure to O22 produces produces ‘Unready’ state in which O‘Unready’ state in which O22
is not fully reducedis not fully reduced..
But if electrons are readily But if electrons are readily available, Oavailable, O22 is reduced to is reduced to
‘water’‘water’
and ‘Ready’ state is formed.and ‘Ready’ state is formed.
NiII FeII + O2 + 4e- + 3H+ NiIII-O-FeII + H2O
NiII FeII + O2 + 2e- + H+ NiIII FeII
H
‘Ready’ (Ni-B)
‘Unready’ (Ni-A)(Blocked)
[O]H2O+
trapped O-atom species
The difference between Ni-A (Unready) and Ni-B (Ready)
Ni-B (‘Ready’)nearly pure state
Ni-A or Ni-SU (‘Unready’)
New crystallographic refinement leads tore-interpretation of the earlier structure of enzyme crystallised mainly in the ‘Unready’ state (Fontecilla-Camps).
0.00
0.20
0.40
0.60
0.80
1.00
1800 2300 2800 3300 3800 4300 4800 5300 5800
Nor
mal
ized
cu
rren
t
Time / sec
-8 mV
-158 mV
-58 mV
-33 mV
-78 mV
Reductive activation of Unready.As reductive step potential is raised, rateof activation slows down.
Excellent first-order traces in all cases
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
-0.3 -0.1 0.1 0.3
Actual DatapH 6
Best Fit
Actual DatapH 7
Best Fit pH7
Rate / s-1
Potential / V vs SHE
pH 6.0
pH 7.0
Plot reveals sharp potential dependence of rate ofactivation of Unready state (5-100% H2)
limiting rate reachedat -100 mV (pH 6)
Time /sec.
1500 2500 35003000 40002000
Nor
mal
ized
cur
rent
0.0
0.2
0.4
0.6
0.8
1.0
H2 introduced after 900s at -158 mV
H2 present from start of reductive step
242 mV, N2
-158 mV, N2
900 s
Time /sec.
1500 2500 35003000 40002000
Time /sec.
1500 2500 35003000 40002000
Nor
mal
ized
cur
rent
0.0
0.2
0.4
0.6
0.8
1.0
H2 introduced after 900s at -158 mV
H2 present from start of reductive step
242 mV, N2
-158 mV, N2
900 s
Nor
mal
ized
cur
rent
0.0
0.2
0.4
0.6
0.8
1.0
H2 introduced after 900s at -158 mV
H2 present from start of reductive step
242 mV, N2
-158 mV, N2
900 s
Does Unready form of hydrogenase activate without H2 ?
current
Measure extent of ‘activation’ under N2 as function of potential
0.00
0.25
0.50
0.75
1.00
0 1000 2000 3000 4000
i(A)
Inject O2
-228 mV
-158 mV
242 mV, N2
-228 mV/ -158 mV
H2-558 mV, N2
H2 oxidationcurrent
time / s
Time Under N2 / sec
% A
ctiv
ity
Rec
over
ed =
Unr
ead
y / U
nrea
dy +
Act
ive
Enz
yme -228 mV
-158 mV
-78 mV
Time Under N2 / sec
% A
ctiv
ity
Rec
over
ed =
Unr
ead
y / U
nrea
dy +
Act
ive
Enz
yme -228 mV
-158 mV
-78 mV
0
20
40
60
80
100
120
0 1000 2000 3000 4000
In absence of H2, reduction of Unready proceedsto a position of equilibrium
Lamle et al. in preparation