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