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

$$ NSF $$$$ TSR&TP $$$$ ECS $$