High-temperature electrocatalysis using thermophilic P450 CYP119: Dehalogenation of CCl4 to CH4

Article abstract of DOI:10.1021/ja0488333

The use of a thermophilic cytochrome P450, CYP119, in electrocatalytic dehalogenations of C₁ halocarbon solvents is studied. Temperature stable enzyme-modified electrodes were constructed using sol-gel and polymeric surfactant approaches. CYP119 deposited in a dimethyldidodecylammonium poly(p-styrene sulfonate) (DDAPSS) film has good retention of electrochemical activity up to 80 °C. At potentials approaching the Fe^II/I couple, the CYP119/DDAPSS films demonstrate high catalytic dehalogenations of the C₁ chloromethanes CCl₄, CHCl₃, and CH₂Cl₂. Product analysis identified mixtures of sequentially dechlorinated products up to methane; no evidence for radical-coupled products was observed. The yield of methane from the CYP119-catalyzed reduction of CCl₄ is increased 35-fold from 25 °C to 55 °C. In combination with the lack of C₂ products, the facility of an overall eight-electron reductive dehalogenation suggests that the substrate is constrained within the protein during electrocatalytic turnover. Copyright

Full text of DOI:10.1021/ja0488333

Published on Web 06/24/2004
High-Temperature Electrocatalysis Using Thermophilic P450 CYP119:
Dehalogenation of CCl4 to CH4
Emek Blair, John Greaves, and Patrick J. Farmer*
Department of Chemistry, UniVersity of California, IrVine, California 92697-2025
Received March 1, 2004; E-mail: pfarmer@uci.edu
Chloro-organics, such as chloroform and methylene chloride, are
produced on the hundreds of millions of pounds per year scale.1
These hepatotoxic and carcinogenic solvents2 have been released
into the environment causing stratospheric ozone depletion3,4 and
contamination of agricultural land. The widespread use of chlori-
nated solvents has resulted in their presence at most of the hazardous
waste sites on the EPA’s national priority list,5 and carbon
tetrachloride has specifically been banned from industrial use in
many nations by the Montreal Protocol, effective January 1, 1996.6
Carbon tetrachloride is still generated as a major byproduct during
chloroform and methylene chloride production. Bioremediation has
had only limited success as the dechlorination turnover is inhibited
Figure 1. Comparison of the FeIII/II anodic currents at various temperatures
by increasing concentrations of CCl4.7
for CYP119 modified electrodes for identical loadings: solid bars denote
CYP119/DDAPSS in 100 mM pH 6 iP buffer; striped bars denote CYP119/
sol-gel/DDAB in 50 mM pH 4 iP buffer. Conditions: 500 mV/s, Pt counter
electrode, Ag/AgCl reference electrode.
Dehalogenations utilizing chemical reductants and biocatalysts,
such as cobalamin (Co), coenzyme F430 (Ni), and P450cam (Fe),
have been demonstrated for various chlorinated toxins.8-11 Likewise,
several groups have studied electrocatalytic dehalogenation using
heme protein or porphyrin electrocatalysts.12-16 However, complete
dehalogenation of polychlorinated substrates is difficult for the C1
halocarbon solvents. In this communication, we describe efficient
multiple dehalogenations of C1 halocarbons at elevated tempera-
tures, electrochemically catalyzed by a thermophilic enzyme.
Extremophilic enzymes are of wide interest because of their
stability at extremes of temperature, pressure, and pH which may
enable their use under harsh industrial applications. Cytochrome
P450 CYP119 was discovered in the genome of Sulfolobus
solfataricus, an extremophilic archaebacteria found in sulfurous
volcanic hot springs. CYP119 has an unusually high denaturation
point of ca. 90 °C and tolerance for extreme pHs and pressures for
extended periods in solution.17-21 We have previously demonstrated
that CYP119 gives excellent electrochemical response when im-
mobilized in a dimethyldidodecylammonium bromide (DDAB) film
on a pyrolitic graphite electrode (PG)20 and have utilized it for the
catalytic reduction of NOx species such as nitrite and nitric oxide.22
To determine the effect of temperature on the electrochemical
activity of CYP119, thermostable immobilization schemes using
sol-gels and polymer-based surfactants were investigated. P450cam,
immobilized in a methyltriethoxysilane sol-gel film on graphite
as an electrochemical sensor for camphor and pyrene, retains
prolonged stability in organic solvents.23 Likewise, alkylammonium
poly(p-styrene sulfonate) are thermostable and insoluble in aqueous
and organic solutions making them suitable for a varied array of
electrochemical environments;24 the combination of dimethyl-
didodecylammonium poly(p-styrene sulfonate), or DDAPSS, to
protein voltammetry was recently demonstrated for myoglobin.13
As determined electrochemically, the CYP119/sol-gel film
remained stable until ca. 60 °C (S1, Supporting Information), but
the anodic current drops off at higher temperatures to ca. 45% of
that at 30 °C, Figure 1.
Figure 2. Electrocatalytic reductive dehalogenations of C1 chlorocarbon
substrates at room temperature. (Top) CYP119/DDAPSS in the presence
of 10 mM CH2Cl2 (solid), CHCl3 (dashed), and CCl4 (dotted). (Bottom)
Voltammogram of catalyst in absence of substrate. Conditions: 100 mM
pH 6 iP buffer, 20 mV/s, and Pt counter electrode, Ag/AgCl reference.
observed between 80 °C and 90 °C. Variable temperature absorption
measurements demonstrate that the heme Soret band does not shift
with temperature in either system (S2B, Supporting Information).
Over the accessible temperature range, observed E1/2 for the FeIII/II
couple decreases linearly with increased temperature, from -213
mV at 25 °C to -278 mV at 80 °C, ∆E1/2 ) -1.07 × 10-3 V/°C
(S4, Supporting Information). This represents a somewhat larger
temperature shift than those seen for other thermophilic and
nonthermophilic redox proteins25,26 but may contain contributions
from the film dielectric.
At room temperature, the presence of C1 chlorinated substrates
cause large increases in cathodic current in voltammograms of
CYP119/DDAPSS, indicative of catalytic reductions of these
compounds, Figure 2. The catalytic currents increase linearly with
substrate concentrations well beyond the substrates solubility in
water, indicative of preconcentration of the hydrophobic compounds
within the film (S5, Supporting Information). GC-MS analysis of
At 80 °C, CYP119/DDAPSS films retain 93% of the current at
the FeIII/II couple at 30 °C, and only a moderate loss of signal is
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10.1021/ja0488333 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
strated as a superior catalyst for the reduction of CCl4 to CH4, an
overall eight-electron reduction. The advantage over other catalysts
may lie within the protein structure itself, i.e., that the rigidity and
stability of the thermophilic protein traps the substrate within the
active pocket, facilitating multiple reductions of individual sub-
strates.
Acknowledgment. We are indebted to Professor Tom Poulos
and Jason Yano at University of California, Irvine for their
assistance in expression of the CYP119 used in this work. We also
thank Professor Don Blake for assistance with the GC analysis.
This research is supported by the National Science Foundation (PJF
CHE-0100774). E.B. acknowledges a graduate fellowship from the
UC TSR&TP.
Figure 3. Effect of temperature on catalytic dehalogenation of CCl4. (Top)
Linear sweep voltammograms of CYP119/DDAPSS in the presence of
saturated CCl4 at (a) 25 °C, (b) 55 °C, (c) 75 °C. (Bottom) Voltammogram
of CYP119/DDAPSS at 200 mV/s. (Right) Volume of methane produced
during electrocatalysis of CCl4 at 25 °C (× 10) and 55 °C after electrolysis
for 20 min at -1150 mV, average of three trials. Solid bars denote CYP119
catalysis, and striped are control experiments in absence of CYP119.
Supporting Information Available: Experimental details, including
characterizations of the protein-modified electrodes, temperature de-
pendence of the FeIII/II couple, methods of analysis of the products of
bulk electrolysis, control experiments, and a comparison of nitrite
reduction at elevated temperatures; seven pages. This material is
available free of charge via the Internet at http://pubs.acs.org.
products resulting from bulk electrolysis identified sequentially
dechlorinated C1 products but gave no evidence of C2 products,
e.g., chloroethanes (S6, Supporting Information). The electron
turnover per protein at equivalent concentrations of substrate are
52.1 s-1 for CCl4, 27.5 s-1 for CHCl3, and 4.5 s-1 for CH2Cl2,
following the differences in electron affinity of the substrates.27
The catalytic waves occur close to the FeII/I couple, suggesting that
the FeI state is the active catalyst and that the reactions proceed
via an overall two-electron reduction process, eq 1.
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