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ELECTROCATALYSIS BY ELECTROCATALYSIS BY THERMOPHILIC CYTOCHROME THERMOPHILIC CYTOCHROME
P450 CYP119 IN SURFACE-MODIFIED P450 CYP119 IN SURFACE-MODIFIED ELECTRODESELECTRODES
Emek Blair and Patrick J. Farmer
Department of Chemistry University of California, Irvine
516 Rowland HallIrvine, CA 92697
OverviewOverview Background descriptionBackground description Electrochemical characterization of P450 Electrochemical characterization of P450
CYP119CYP119 Show stability of thin films at elevated Show stability of thin films at elevated
temperaturestemperatures Catalysis of CCatalysis of C11 Cl-R vs. temperature Cl-R vs. temperature
Elute best conditions to fully dechlorinate CElute best conditions to fully dechlorinate C2 2 Cl-RCl-R pH and temperature pH and temperature
Number of Number of California EPA California EPA Sites where Sites where
Chlorinated VOCs Chlorinated VOCs are Priority are Priority PollutantsPollutants
Priority 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/.
Chlorinated Chlorinated VOCsVOCs
Hepatotoxic (bio half Hepatotoxic (bio half life is 3-7 days)life is 3-7 days)
Depletes ozone Depletes ozone Produced on the billion Produced on the billion
of lb./year scaleof lb./year scale increased increased
concentrations found concentrations found in environment in environment
CClCCl44 is banned from is banned from industrial use (Montreal industrial use (Montreal Protocol 1/1/96)Protocol 1/1/96)
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, 2000Rusling, JACS, 1993
Wacket, Env. Sci. Tech., 1991Krone, Biochem., 1989 Wackett, Biochem., 1993
Freedman, Env. Sci. Tech., 1995
P450 CYP119P450 CYP119 Heme enzyme from Heme enzyme from
Sulfolbus solfataricusSulfolbus solfataricus Found in thermal Found in thermal
vents vents ThermophilicThermophilic
Melting temperature Melting temperature ~90ºC~90ºC
AcidophilicAcidophilic Optimal growing pH Optimal growing pH
~3.5 ~3.5
Traditionally inaccessible environments may be usedYano, J., J. Biol. Chem. 2000
thin filmca. 1 micronPyrolytic
Graphiteelectrode
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
P450P450
Fe
only 4 of >300 layers are shown.
NCH3
CH3C12H25
C12H25
-Br
0 -300 -600 -900 -1200
-1.0
-0.5
0.0
0.5
1.0
Cur
rent
(A
)
Potential (mV)
FeIII/II FeII/I
Rusling 1998
0 -200 -400 -600 -800 -1000-9
-6
-3
0
3
6
9
12
Cur
rent
(A
)
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 CYP119 Electroactivity in Sol-gel/DDAB FilmsFilms
DisadvantagesDisadvantages weak signalweak signal only Feonly Fe+3/+2+3/+2 observed observed catalysis Cl-R not catalysis Cl-R not
observedobserved
CYP119 in Surfactant Films (DDAPSS)CYP119 in Surfactant Films (DDAPSS)
200 0 -200 -400 -600 -800 -1000 -1200
-6-4-202468
10
Cur
rent
(A
)
Potential (mV vs Ag/AgCl)
Conditions: Scan rate = 500 mV/s, pH 7, 100mM iP buffer
20ºC
50ºC
70ºC
0 500 1000 1500 20000.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
Cur
rent
(A
)
Scan Rate (mV/s)
NCH3
CH3C12H25
C12H25
O3S CH
CH2
n
20ºC
60ºC
s = 7.2*10-6 C/V
s = 6.27*10-6 C/Vfilm loosens with temperature
p-Poly styrene sulfonateDDA
20 30 40 50 60 70 80
-280
-270
-260
-250
-240
-230
-220
-210
Eo (m
V v
s A
g/A
gCl)
Temperature (oC)
ThermodynamicsThermodynamicsG = -nfE = H - TS
-nFE / T = H/ T - S S = nF E/ T
E/ T = -1.068 * 10-3 V/KS = -103.14 eu
E1/2 of CYP119/DDAPSS Fe+3/+2 vs. temperature
Thermophiles -60euNon-thermophiles -10euInorganic redox molecules -5 to 20eu
Dielectric of solvent decreases with temperature stabilizing less charged state
Smith, Anal. Biochem. 1995
Electroactivity of CYP119 FilmsElectroactivity of CYP119 Films
Charge of FeIII/II couple at various temperatures, relative to activity at 30 0C.
Koo, J. of Biol. Chem. 2000,
Black bars: active heme chromophores, and white: residual styrene epoxidation standardized to 20C
E-chem results consistent with solution based experimentsSurfactant stabilizes protein: can heat over 90 oC
30 40 50 60 70 800.00
0.05
0.10
0.15
0.20
0.25 DDAPSS Sol-gel /DDAB
Cha
rge
(mC
)
Temperature (oC)
92%
43%
Reduction of CReduction of C11 Chlorocarbons ChlorocarbonsCYP119/DDAPSS films in pH 6 buffer at 20 mV/s scan rate
CYP119 capable of dechlorinating C1 chlorocarbons
0 -300 -600 -900 -1200-0.250.000.25
Cur
rent
(A
)
Potential (mV vs Ag/AgCl)
0.0
1.0
2.0
3.0CCl4
CHCl3
CH2Cl2
CCl4 CHCl3 CH2Cl2Turnover per e- (1/s) 52.1 27.5 4.5Turnover per Cl- (1/s) 26.1 13.8 2.2
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 1400
20
40
60
80
100
x10Perc
ent
m/z
11 12 13 14 15 16 17 18 190
20
40
60
80
100
. Per
cent
m/z11 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
H2H+ + 2e-
-Cl-
2H+ + 2e-
-Cl-
2H+ + 2e-
-Cl-
2H+ + 2e-
-Cl-
CYP119-DDAPSS Catalytic Activity CYP119-DDAPSS Catalytic Activity vs Temperaturevs Temperature
Temperature Effect Temperature Effect on Substrateon Substrate increase solubilityincrease solubility increase diffusion increase diffusion
raterate increase rate of increase rate of
degradationdegradation increase catalysis increase catalysis
potentialpotentialCatalysis 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
Cur
rent
(mA
)
200 0 -200 -400 -600 -800 -1000 -1200 -1400
-2.0
0.0
2.0
Potential (mV vs Ag/AgCl)
(A
)
75ºCSaturatedCCl4
55ºC
25ºC
25ºC
CYP119/DDAPSS CHCYP119/DDAPSS CH4 4 ProductionProduction
Solid: CYP119/DDAPSSStriped: 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°C25
0.0
0.5
1.0
1.5
2.0
Met
hane
Pro
duce
d (
L)
Temperature (oC)55
0
20
40
60
ThermophileThermophile P450 CYP119 is electrochemically P450 CYP119 is electrochemically
stable in thin films over all water stable in thin films over all water temperatures temperatures
8e8e-- reduction of CCl reduction of CCl44 Temperature influences degradation Temperature influences degradation
rates and productsrates and productspH 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
ClH
H
H
Cl
Cl
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
250 0 -250 -500 -750 -1000
-1.0
-0.5
0.0
0.5
1.0
Cur
rent
(A
)
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 Fe of CYP/DDAPSS FeIII/IIIII/II vs. pH vs. pH
2 4 6 8 10 12 14
-600
-500
-400
-300
-200
-100
Pot
entia
l (m
V)
pH
ca. -50 mV/ pH unit
0 -350 -700 -1050 -1400
-1
0
1
2
Cur
rent
(A
)
Potential (mV vs. Ag/AgCl)4 6 8 10 12 14
-1160
-1140
-1120
-1100
-1080
-1060
Pot
entia
l (m
V)
pH
(5-coordinate) FeII FeI (4 coordinate)
EE1/21/2 of CYP/DDAPSS Fe of CYP/DDAPSS FeII/III/I vs. pH vs. pH
As pH increases CCl4 reduction potential decreasesCCl4 + 8e- + 4H+ CH4 + 4Cl-
ca. -10 mV/ pH unit
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 strongcatalysis (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 oC
55 oC
Cur
rent
(A
)
25 oC
Potential (mV vs. Ag/AgCl)
0 -300 -600 -900 -1200-3
0
3
6
9-10
0
10
20
30
40
25 oCCur
rent
(A
)
Potential (mV)
55 oC
pH 7 pH 10
Products
020406080
020406080
020406080
pH 7 pH 10
25 0C-1150 mV
55 0C-1150 mV
55 0C-700 mV
% o
f Tot
al G
as
Cl
ClCl
Cl
Cl
Cl
ClCl
Cl
pH 10 and lower E is more efficient at total dechlorination
-while pH 7 at 55 oC (-700 mV) has highest dechlorination efficiency, largest overal ethane production at pH 10 at 55 oC (-1150 mV)
ConclusionsConclusions P450 CYP119 is electrochemically stable in thin films P450 CYP119 is electrochemically stable in thin films
over all water temperatures over all water temperatures 8e8e-- reduction of CCl reduction of CCl4 4 and 6eand 6e-- reduction of MeCCl reduction of MeCCl3 3 Temperature and pH influences degradation rates Temperature and pH influences degradation rates
and productsand products
Understand dechlorination mechanismUnderstand dechlorination mechanism Look at oxygenationLook at oxygenation
toxicologytoxicology
Future WorkFuture Work
AcknowledgmentsAcknowledgmentsFarmer Research
Group
$$ NSF $$$$ TSR&TP $$$$ ECS $$