A structured lipid system surrounding the enzymes which is stable at high temperature and acidic conditions is used to characterize P450 CYP119. These mimic the natural lipid bilayers that the enzymes naturally are stable in.

1. ELECTROCATALYSIS BYELECTROCATALYSIS BY
THERMOPHILIC CYTOCHROMETHERMOPHILIC CYTOCHROME
P450 CYP119 IN SURFACE-MODIFIEDP450 CYP119 IN SURFACE-MODIFIED
ELECTRODESELECTRODES
Emek Blair and Patrick J. Farmer
Department of Chemistry
University of California, Irvine
516 Rowland Hall
Irvine, CA 92697

2. OverviewOverview
 Background descriptionBackground description
 Electrochemical characterization of P450 CYP119Electrochemical characterization of P450 CYP119
 Show stability of thin films at elevated temperaturesShow stability of thin films at elevated temperatures
 Catalysis of CCatalysis of C11 Cl-R vs. temperatureCl-R vs. temperature
 Elute best conditions to fully dechlorinate CElute best conditions to fully dechlorinate C22 Cl-RCl-R
 pH and temperaturepH and temperature

3. Number ofNumber of
California EPA SitesCalifornia EPA Sites
where Chlorinatedwhere Chlorinated
VOCs are PriorityVOCs are Priority
PollutantsPollutantsPriority Pollutant Contaminated
Sites (of 96)
1,1,1 -trichloroethane 46
trichloroethylene 34
tetrachloroethylene 28
dichloroethylene 51
vinyl chloride 31
pentachloroethane 1
carbon tetrachloride
chloroform
18
42
methylene chloride 20
Chlorinated VOCs 68
U.S. EPA National Priorities List, 2 October
2002, www.epa.gov/superfund/sites/npl/.
ChlorinatedChlorinated
VOCsVOCs
 Hepatotoxic (bio half lifeHepatotoxic (bio half life
is 3-7 days)is 3-7 days)
 Depletes ozoneDepletes ozone
 Produced on the billion ofProduced on the billion of
lb./year scalelb./year scale
 increasedincreased
concentrations found inconcentrations found in
environmentenvironment
 CClCCl44 is banned fromis banned from
industrial use (Montrealindustrial use (Montreal
Protocol 1/1/96)Protocol 1/1/96)

4. Dechlorination
ElectrocatalysisSolutionBioremediation
CCl4 inhibits
protein catalysis
Yields:
CO, CO2, CS2
Vit B12 (Co)
coenzyme F430 (Ni)
heme (Fe)
Yields:
Cl products
P450cam
Mb
Yields:
Cl products or
not characterized
Rivera, JACS, 2000
Rusling, JACS, 1993
Wacket, Env. Sci. Tech., 1991
Krone, Biochem., 1989
Wackett, Biochem., 1993
Freedman, Env. Sci. Tech., 1995

5. P450 CYP119P450 CYP119
 Heme enzyme fromHeme enzyme from
Sulfolbus solfataricusSulfolbus solfataricus
 Found in thermalFound in thermal
ventsvents
 ThermophilicThermophilic
 Melting temperatureMelting temperature
~90ºC~90ºC
 AcidophilicAcidophilic
 Optimal growing pHOptimal growing pH
~3.5~3.5
Traditionally inaccessible environments may be used
Yano, J., J. Biol. Chem. 2000

6. thin film
ca. 1 micronPyrolytic
Graphite
electrode
Purified P450 +
ddab
•P450 cast in thin films of DimethylDidodecyl
Ammonium Bromide (DDAB) on basal plane graphite
gives improved electrochemical response.
Protein - Surfactant FilmsProtein - Surfactant Films
P450
Fe
Fe
P450
P450
Fe
only 4 of >300 layers are shown.
N
CH3
CH3
C12H25
C12H25
-
Br
0 -300 -600 -900 -1200
-1.0
-0.5
0.0
0.5
1.0
Current(µA)
Potential (mV)
FeIII/II
FeII/I
Rusling 1998

7. 0 -200 -400 -600 -800 -1000
-9
-6
-3
0
3
6
9
12
Current(µA)
Potential (mV vs Ag/AgCl)
Cyclic voltammograms of CYP119 sol gel films: Conditions: pH 4, 50 mM
iP buffer with 20 mM KCl as electrolyte, 500 mV/s
20ºC
55ºC
90ºC
CYP119 Electroactivity in Sol-gel/DDAB FilmsCYP119 Electroactivity in Sol-gel/DDAB Films
 DisadvantagesDisadvantages
 weak signalweak signal
 only Feonly Fe+3/+2+3/+2
observedobserved
 catalysis Cl-R notcatalysis Cl-R not
observedobserved

8. CYP119 in Surfactant Films (DDAPSS)CYP119 in Surfactant Films (DDAPSS)
200 0 -200 -400 -600 -800 -1000 -1200
-6
-4
-2
0
2
4
6
8
10
Current(µA)
Potential (mV vs Ag/AgCl)
Conditions: Scan rate = 500 mV/s,
pH 7, 100mM iP buffer
20ºC
50ºC
70ºC
0 500 1000 1500 2000
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
Current(µA)
Scan Rate (mV/s)
N
CH3
CH3
C12H25
C12H25
O3S CH
CH2
n
20ºC
60ºC
s = 7.2*10-6
C/V
s = 6.27*10-6
C/V
film loosens with temperature
p-Poly styrene sulfonateDDA

9. 20 30 40 50 60 70 80
-280
-270
-260
-250
-240
-230
-220
-210
E
o
(mVvsAg/AgCl)
Temperature (
o
C)
ThermodynamicsThermodynamics
∆G = -nfE = ∆H - T∆S
-nF∆E / ∆T = ∆H/ ∆T - ∆S
∆S = nF ∆E/ ∆T
∆E/ ∆T = -1.068 * 10-3
V/K
∆S = -103.14 eu
E1/2 of CYP119/DDAPSS Fe+3/+2
vs.
temperature
Thermophiles -60eu
Non-thermophiles -10eu
Inorganic redox molecules -5 to 20eu
Dielectric of solvent decreases with temperature stabilizing less
charged state
Smith, Anal. Biochem. 1995

10. Electroactivity of CYP119 FilmsElectroactivity of CYP119 Films
Charge of FeIII/II
couple at various
temperatures, relative to activity at 30
0
C.
Koo, J. of Biol. Chem. 2000,
Black bars: active heme chromophores,
and white: residual styrene epoxidation
standardized to 20°C
E-chem results consistent with solution based experiments
Surfactant stabilizes protein: can heat over 90 o
C
30 40 50 60 70 80
0.00
0.05
0.10
0.15
0.20
0.25
DDAPSS
Sol-gel /DDAB
Charge(mC)
Temperature (
o
C)
92%
43%

11. Reduction of CReduction of C11 ChlorocarbonsChlorocarbons
CYP119/DDAPSS films in pH 6 buffer at 20 mV/s scan rate
CYP119 capable of dechlorinating C1 chlorocarbons
0 -300 -600 -900 -1200
-0.25
0.00
0.25
Current(µA)
Potential (mV vs Ag/AgCl)
0.0
1.0
2.0
3.0
CCl4
CHCl3
CH2Cl2
CCl4 CHCl3 CH2Cl2
Turnover per e-
(1/s) 52.1 27.5 4.5
Turnover per Cl-
(1/s) 26.1 13.8 2.2

12. Overall ReactionOverall Reaction
P450 CYP119 is capable of totally dechlorinating
carbon tetrachloride via 8e-
reduction
Mass-spectrometry detected progressive dechlorination CCl4
0 20 40 60 80 100 120 140
0
20
40
60
80
100
x10
Percent
m/z
11 12 13 14 15 16 17 18 19
0
20
40
60
80
100
.Percent
m/z
11 12 13 14 15 16 17 18 19
0
20
40
60
80
100
.
m/z
CHCl3
CD2H2CDH3
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
H
H
Cl
Cl
H
H
Cl
H
H
H
H
H
2H+
+ 2e-
-Cl-
2H+
+ 2e-
-Cl-
2H+
+ 2e-
-Cl-
2H+
+ 2e-
-Cl-

13. CYP119-DDAPSS Catalytic Activity vsCYP119-DDAPSS Catalytic Activity vs
TemperatureTemperature
 Temperature Effect onTemperature Effect on
SubstrateSubstrate
 increase solubilityincrease solubility
 increase diffusion rateincrease diffusion rate
 increase rate ofincrease rate of
degradationdegradation
 increase catalysisincrease catalysis
potentialpotential
Catalysis increases but
is still substrate-limited200 mV/s scan rate, pH 6, 100 mM iP buffer
0.0
0.5
1.0
1.5
2.0
Current(mA)
200 0 -200 -400 -600 -800 -1000 -1200 -1400
-2.0
0.0
2.0
Potential (mV vs Ag/AgCl)
(µA)
75ºCSaturated
CCl4
55ºC
25ºC
25ºC

14. CYP119/DDAPSS CHCYP119/DDAPSS CH44 ProductionProduction
Solid: CYP119/DDAPSS
Striped: DDAPSS
20 min catalysis at -1150 mV
Rate doubles → methane
production increases >32
times
Energy barrier of dechlorination overcome by temperature.
GC-FID
55°C
25°C
25
0.0
0.5
1.0
1.5
2.0
MethaneProduced(µL)
Temperature (
o
C)
55
0
20
40
60

15. ThermophileThermophile
 P450 CYP119 is electrochemically stable inP450 CYP119 is electrochemically stable in
thin films over all water temperaturesthin films over all water temperatures
 8e8e--
reduction of CClreduction of CCl44
 Temperature influences degradation ratesTemperature influences degradation rates
and productsand products
pH Stable ToopH Stable Too
 Determine how to control degradation pathwaysDetermine how to control degradation pathways
 varying pHvarying pH
 varying temperaturevarying temperature
H
H
H
Cl
Cl
Cl
H
H
H
Cl
Cl
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H

16. 250 0 -250 -500 -750 -1000
-1.0
-0.5
0.0
0.5
1.0
Current(µA)
Potential (mV vs. Ag/AgCl)
Films scanned at 100 mV/s in 25 mM iP solution. PG working electrode, 3M KCl Ag/AgCl
reference electrode, Pt wire auxiliary electrode, and a glass calomel combination pH micro-
electrode
2.82, 7.02, 13.56
EE1/21/2 of CYP/DDAPSS Feof CYP/DDAPSS FeIII/IIIII/II
vs. pHvs. pH
2 4 6 8 10 12 14
-600
-500
-400
-300
-200
-100
Potential(mV)
pH
ca. -50 mV/ pH unit

17. 0 -350 -700 -1050 -1400
-1
0
1
2
Current(µA)
Potential (mV vs. Ag/AgCl)
4 6 8 10 12 14
-1160
-1140
-1120
-1100
-1080
-1060
Potential(mV)
pH
(5-coordinate) FeII
↔ FeI (
4 coordinate)
EE1/21/2 of CYP/DDAPSS Feof CYP/DDAPSS FeII/III/I
vs. pHvs. pH
As pH increases CCl4 reduction potential decreases
CCl4 + 8e-
+ 4H+
→ CH4 + 4Cl-
ca. -10 mV/ pH unit

18. 1,1,1-trichloroethane Reduction1,1,1-trichloroethane Reduction
50 mM pH 7 iP buffer: red) buffer
solution black) sat 1,1,1-trichloroethane
E2 Catalysis rate increases with
temperature (4, 14, 22 s-1
)
- E1 also exhibits strong
catalysis (loss of reversibility)
Ultimately: This is similar behavior to CCl4 reduction
0 -300 -600 -900 -1200
0
10
20
30
40
50
0 -300 -600
85
o
C
55
o
C
Current(µA)
25
o
C
Potential (mV vs. Ag/AgCl)

19. 0 -300 -600 -900 -1200
-3
0
3
6
9
-10
0
10
20
30
40
25 oC
Current(µA)
Potential (mV)
55 oC
pH 7
pH 10
Products
0
20
40
60
80
0
20
40
60
80
0
20
40
60
80
pH 7 pH 10
25 0C
-1150 mV
55 0C
-1150 mV
55 0C
-700 mV
%ofTotalGas
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
pH 10 and lower E is more efficient at
total dechlorination
-while pH 7 at 55 o
C (-700 mV)
has highest dechlorination efficiency,
largest overal ethane production at pH
10 at 55 o
C (-1150 mV)

20. ConclusionsConclusions
 P450 CYP119 is electrochemically stable in thin filmsP450 CYP119 is electrochemically stable in thin films
over all water temperaturesover all water temperatures
 8e8e--
reduction of CClreduction of CCl44 and 6eand 6e--
reduction of MeCClreduction of MeCCl33
 Temperature and pH influences degradation ratesTemperature and pH influences degradation rates
and productsand products
 Understand dechlorination mechanismUnderstand dechlorination mechanism
 Look at oxygenationLook at oxygenation
 toxicologytoxicology
Future WorkFuture Work

21. AcknowledgmentsAcknowledgments
Farmer Research Group
$$ NSF $$
$$ TSR&TP $$
$$ ECS $$