Chemical modification of cytochrome C improves their catalytic properties in oxidation of polycyclic aromatic hydrocarbons

ELSEVIER

Chemical modification of cytochrome C improves their catalytic properties in oxidation of polycyclic aromatic hydrocarbons Raunel Tinoco and Rafael Vazquez-Duhalt Institute

de Biotecnologia-UNAM,

Cuernavaca,

Morelos,

Mexico

Free amino and carboxylic groups of the horse heart cytochrome C were modified by chemical reaction with methyl, trimethylsilyl (TMS), and poly(ethylene)glycol (PEG) moieties. As a consequence of chemical modification, the heme environment (active site) was altered. The kinetic constants and substrate specificities were determined for the differently modified cytochromes. A cytochrome with a double modification (PEG on free amino groups and methyl esters on carboxylic groups) was able to oxidize 17 aromatic compounds from 20 tested while the unmodified protein was only able to oxidize 8 compounds. This work shows that chemical modification 0 1998 of a biocatalyst could be a tool for the design of a new biocatalyst with environmental purposes. Elsevier Science Inc. Keywords:

Biocatalysis;

chemical

modification;

cytochrome;

Introduction Polycyclic aromatic hydrocarbons (PAHs) are considered to be a potential health risk because of their possible carcinogenic and mutagenic activities.’ PAHs are components of coal tar, creosote, and crude oil and are formed by the incomplete combustion of organic material. During the last century, oil has been used extensively. It has been the origin of some widespread PAH pollution. Our work has been focused on the biocatalytic oxidation of aromatic compounds mainly using hemoproteins.2-6 Enzymes such as lignin peroxidase3.7s and cytochrome P 450 9-‘o which contain a heme prosthetic group are able to oxidize PAH in vitro. Biocatalytic oxidations of different aromatic compounds with nonenzymatic hemoproteins in the presence of hydrogen peroxide have been reported. Thiophenes and organosulfides are transformed into the respective sulfoxides by cytochrome C.* Dibenzothiophene is oxidized by hemoglobin’ ’ while yeast cytochrome5 and hemoglobin6 are able to form quinones from PAH in the presence of hydrogen peroxide. The products formed by the

enzyme; oxidation;

polycyclic

aromatic

hydrocarbons

PAH oxidation with lignin peroxidase, cytochrome C, and hemoglobin are the same (mainly quinones and hydroxylated compounds). This suggests the same catalytic mechanism.3x5,6 Cytochrome C could be considered a good biocatalyst because it has some advantages when compared with hemoenzymes; a covalently bonded prosthetic group which is not lost by adding organic solvents, catalytic activity at a high concentration of organic solvents (up to 90% tetrahydrofuran), and activity over a wide pH range (from 2 to 12).‘* Chemical modification of the enzyme surface has been performed to improve the catalytic activity in organic solvents.‘3-‘5 In addition to amphipathic groups such as poly(ethylene)glycol (PEG), aromatic moieties have been covalently bonded on lignin peroxidase to alter their catalytic properties.4 In this work, horse heart cytochrome C was modified by chemical reactions on the protein surface and the active-site (prosthetic group). Their effects on biocatalytic properties were determined.

Materials and methods Chemicals Address reprint requests to Dr. Rafael Vazquez-Duhalt, Instituto de Biotecnologia-UNAM. Apartado Postal 5 10-3, Cuernavaca, Morelos 62250, Mexico Received 7 February 1997; Revised 19 May 1997; accepted 19 May 1997

Enzyme and Microbial Technology 22%12, 1998 0 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

Horse heart cytochrome C, sodium cyanoborohydrate, formaldehyde, methoxypoly(ethylene)glycol activated with cyanuric chloride, boron trifluoride-methanol (BF,-methanol), and N,o-bis(tri-

0141-0229/98/$19.00

PII s0141-0229(97)00073-2

Chemical

modification

methylsilyl)acetamide (BSA) were purchased from Sigma Chemical Company (St. Louis, MO). Salt buffers were obtained from J. T. Baker (Phillipsburg, NJ) and organic solvents (HPLC grade) were obtained from Fisher Scientific (Fairlawn, NJ). All aromatic substrates were purchased from Aldrich (Milwaukee, WI). The iso-1-cytochrome variants were a gift from Dr. A. Grant Mauk (Department of Biochemistry and Molecular Biology, University of British Columbia).

Chemical modifications Poly(ethylene)glycol-cytochrome c (PEG-Cyt) was obtained according to Gaertner and Puigserver I3 by using activated poly(ethylene)glycol with cyanuric chloride (MW 5,000) as described previously.‘5 Methylated-cytochrome c (Cyt-Met) was obtained by the alkylation of free carboxylic acid groups to form methyl esters. Lyophilized horse heart cytochrome C (6 mg) was dissolved in 2 ml of N’N-dimethyl formamide and then 2 ml of trifluoridemethanol reagent (BF,-methanol) were added and the reaction mixture was held for 12 h at room temperature. The reaction mixture was diluted to 40 ml with phosphate buffer pH 6.1 and filtered through a 0.45 pm nylon membrane. Filtrate was then dialyzed and concentrated on an Amicon ultrafiltration system with a 10,000 Da membrane. Methylated PEG-cytochrome C (PEG-Cyt-Met) was prepared with the same procedure but starting with lyophilized PEG-Cyt. Trimethylsilyl-cytochrome C (CytTMS) was obtained from a reaction with N,o-bis(trimethyJsilyJ)acetamide (BSA). Lyophilized horse heart cytochrome C (9 mg) was dissolved in 2 ml of N’N-dimethylformamide, 0.6 ml of N,o-bis(trimethylsilyI)acetamide (BSA) was then added, and the reaction mixture was shaken for 2 h at room temperature. The reaction mixture was then diluted to 50 ml with phosphate buffer, and dialyzed and concentrated on a ultrafiltration system with a 10,000 Da membrane. The trimethylsilyl derivative of PEGcytochrome C (PEG-Cyt-TMS) was prepared with the same procedure but starting with lyophilized PEG-Cyt. A fully methylated cytochrome was prepared by forming methylesters of carboxylic groups as mentioned above, and methyl derivatives from free amino groups by reductive alkylation with formaldehyde and sodium cyanoborohydrate. ” The degree of modification was determined by measuring the number of free amino groups in the cytochrome molecule with trinitrobenzene sulfonate (TNBS). ”

Reaction conditions The reaction mixture (1 ml) contained 20 FM PAH and from 0.3-5.0 FM cytochrome preparation in 60 mu phosphate buffer pH 6.1 containing 15% acetonitrile. The reactions were performed at room temperature (20-25°C) and started by adding 1 mM hydrogen peroxide. The reaction progress was monitored by a HPLC system (Perkin-Elmer) equipped with a reverse-phase column (150 X 3.9 mm) Resolve C,, 5 pm (Millipore). The decrease in the amounts of aromatic substrate were determined by measuring the decrease in their peak area at A,,, or AZsO with a Turbochrom (Perkin-Elmer) workstation after calibration with standards. The specific activity was estimated by measuring the mol oxidized substrate mol- ’ cytochrome mini ’ It was expressed in units mm _-‘. All reactions were done in triplicate, and the mean and standard deviations are reported. To obtain enough products for their identification, IO-ml reaction mixtures containing 20 FM aromatic compound were treated with cytochrome preparation and hydrogen peroxide. Complete conversion (monitored by HPLC) was obtained by three additions of cytochrome preparation and hydrogen peroxide at l-h intervals. The reaction mixtures were acidified and extracted five times with 2 ml of methylene chloride. The extracts were com-

of cytochrome

C: R. Tinoco and R. Vazquez-Duhalt

bined, dried over Na$O,, and concentrated under N, prior analysis on gas chromatography-mass spectrometry (GC-MS). Kinetic constants of different cytochrome preparations were determined in a medium containing thianthrene and hydrogen peroxide in 15% acetonitrile-phosphate buffer. Two kinetic curves were determined. The first varies the hydrogen peroxide concentration in thianthrene saturation condition and the second varies the thianthrene concentration and hydrogen peroxide saturation conditions. The reaction rate was monitored spectrophotometrically by measuring the decrease in absorbance at 254 nm. The specific activity was estimated by using an extinction coefficient for thianthrene of 35,000 M-‘cm-‘. Inactivation rates were determined by incubating different cytochrome preparations in 1 ml of phosphate buffer pH 6.1 containing I ITIM hydrogen peroxide. After various incubation times, the reaction was started by adding 100 ~1 of pinacyanol in acetonitrile (50 kg ml-‘). The reaction rate was estimated measuring the decrease in absorbance at 603 nm, and the inactivation constant was determined by fitting the data to the first-order equation (A, = A, eek’).

Results and discussion the aim of changing the catalytic behavior of cytochrome C, the molecule was modified by alkylation to form methyl esters. The goal was to modify the propionates of the prosthetic heme group; however, because the methylation is not site specific, all free carboxylic groups could be modified with this technique. The reaction should be performed in organic solvents without the presence of water. Mass transfer limitations during the alkylation reaction can be reduced by having a soluble protein in organic solvents; thus, cytochrome protein was first modified by binding poly(ethylene)glycol (PEG) moieties on the cytochrome surface to obtain a soluble protein in organic solvents. PEG-cytochrome was then used for methylation to form methylated PEG-cytochrome or PEG-Cyt-Met. Oxidation of twenty aromatic compounds (mainly PAHs) were tested by using PEG-Cyt-Met and 1 mM hydrogen peroxide (Table 1). Interestingly by this chemical modification, it was possible to change both the catalytic activity and substrate specificity of the cytochrome. From 20 tested compounds, the PEG-Cyt-Met oxidized 17 while the unmodified cytochrome was only able to oxidize eight aromatic compounds. No differences of products could be found with modified cytochrome when compared with those from unmodified protein. Products from anthacene, pyrene, fluorene, dibenzothiophene, and thianthrene obtained by oxidation with both modified and unmodified cytochrome were identified by GC-MS. The mass spectra matched with those of 9, IO-anthraquinone; 1,8_pyrenodione; 9-fluorenone; dibenzothiophene sulfoxide; and 5,5’-thianthrene sulfoxide, respectively. These products are the same as those found in the oxidation with lignin peroxidase and hemoglobin.‘~“~7 Difference spectra between PEG-Cyt and PEG-Cyt-Met preparations (Figure I) showed a change in the Soret band. This suggested a modification of the ferriporphyrin environment. Chemical modification of the heme group has been performed on horseradish peroxidase’8 and cytochrome Ph5a, I9 leading to a change in the catalytic activity of both enzymes. 8-Formylheme derivatives of horseradish With

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1998, vol. 22, January

9

Papers Table 1 Oxidation of polycyclic aromatic hydrocarbon by unmodified- and methylated polyfethylene)glycol-modified-cytochrome C Specific activity (min’) Aromatic

Unmodified

compound

7,12-Dimethylbenzanthracene 1,2:3,4_Dibenzanthracene Azulene 3-Methylcholanthrene 7-Methylbenzo(a)pyrene 1,2:5,6_Dibenzanthracene Triphenylene Dibenzothiophene Anthracene Thianthrene Pyrene Fluoranthene Acenaphthene Benzofajpyrene Fluorene Phenanthrene Chrysene 9,10-Dimethylanthracene Naphthalene Biphenyl No reaction detected,

24.59 (? 1.52) 2.26;: 0.29) 1.88 (2 0.07) NR NR 0.67y: 0.33 (5 0.49 (k 0.51 (” NR NR 0.22 (2 NR NR NR NR NR NR

0.06) 0.06) 0.06) 0.05)

0.02)

PEG-Cyt-Met 80.33 16.60 14.32 10.96 7.56 5.70 5.27 4.73 3.09 1.41 0.97 0.65 0.40 0.39 0.22 0.17

(” (2 f-c (5 (2 f-’ (2 (2 t-c (2 (” (2 (k (2 (2 (2 NR NR NR NR

3.83) 2.24) 0.57) 0.54) 0.42) 0.31) 1.05) 0.05) 0.32) 0.08) 0.03) 0.09) 0.01) 0.06) 0.01) 0.02)

NR

peroxidase showed different catalytic constants for guaiacol oxidation when compared with native peroxidase.” Replacement of a natural heme group with ferriporphyrin IX dimethyl ester in cytochrome P4s0, increased the affinity for the substrate, thereby suggesting that the methylation of propionate groups of the heme moiety are important in the binding pocket for substrate. l9 In our case, changes in the heme environment could be the origin of the drastic changes in the substrate affinity. In order to determine the role of each modification on the specificity change, four modifications were performed and assayed for oxidation of six selected PAHs. Activated poly(ethylene)glycol (MW 5,000) was used to modify free amino groups located on the protein surface. This modifi-

400

cation has been used to produce soluble enzymes in organic solvents”.*’ or microparticulate suspensions.” PEG modification of cytochrome C has increased the catalytic activity in systems containing organic solvents. ’ * Methyl esterification was performed on free carboxylic acids. The propionate groups of heme could be included as shown in the difference spectra of unmodified and methyl-modified cytochrome (Figure I). Trimethylsilyl (TMS) reagent is able to modify amino, carboxylic, and phenolic groups. All of selected PAHs were oxidized by the PEG-Cyt-Met preparation (Table 2); nevertheless, neither Cyt-Met nor PEG-Cyt were able to oxidize three of six PAHs as unmodified biocatalysts do. As reported previously,” PEG-Cyt showed higher catalytic activity than unmodified cytochrome, but no change in substrate specificity could be detected. TMS modification produces an insoluble protein while PEG-CytTMS is a soluble preparation. This last preparation was able to oxidize fluoranthene, acenaphthene, and chrysene which were not transformed by unmodified cytochrome (data not shown). With TMS modification, bulky groups [-Si(CH,),] could be bonded to heme propionates and accessible amino, carboxylic, and hydroxyl groups, thereby producing a hydrophobic protein; however, steric impediments could be produced. On the other hand, a fully methylated cytochrome was prepared by forming methylesters of carboxylic groups and methyl derivatives from free amino groups by reductive alkylation with formaldehyde. This preparation showed that it had all the free amino groups methylated as determined by TNBS titration. Fully methylated cytochrome showed the same substrate pattern as the unmodified cytochrome, suggesting that PEG modification of amino groups is important for the specificity change. Kinetic constants for these cytochrome preparations were determined (Table 3). The PEG-Cyt preparation showed the highest value of k,,, while PEG-Cyt-TMS showed the lower k,,, value, but differences varied from 70-160’3~ of value found for unmodified cytochrome. In contrast, important differences were obtained on KM values for both substrates, thianthrene and hydrogen peroxide. The PEG modification reduced the KM value (8.8 times for

5

Wovelength (nrr Figure 1 Absorbance spectra (a) and difference spectra (b) of modified and unmodified cytochrome C preparations. Absorbance spectra of oxidized preparations; unmodified cytochrome C (- - - - -), poly(ethylene)glycol-cytochrome k--1, and methylated poly(ethylene)glycol-cytochrome C (- - - - - - -). Difference spectra were obtained by subtraction of absorbance spectrum of modified cytochrome from the unmodified spectrum. Concentration of both preparations was the same on a protein basis; Cyt - PEG-Q-t k--), Cyt - PEG-r&t-Met (- - -1, and PEG-Cyt - PEG-Cyt-Met (- - - - 4

IO

Enzyme Microb. Technol.,

1998, vol. 22, January

Chemical Table 2

Specific activity on polycyclic aromatic

modification

hydrocarbon

of cytochrome

oxidation

with modified

C: R. Tinoco and R. Vazquez-&halt and unmodified

cytochrome

C

Specific activity (min-‘) Aromatic

compound

Pyrene Anthracene Benzo(a)pyrene Fluoranthene Fluorene Phenanthrene Not determined,

W

Cyt-Met

PEG-Cyt

PEG-Q-t-Met

Cyt-TMS

PEG-Cyt-TMS

0.51 0.33 0.22 NR NR NR

0.26 0.35 0.09 NR NR NR

2.78 6.62 5.15 ND NR NR

0.97 3.09 0.39 0.65 0.22 0.17

0.69 0.72 1.40 ND NR NR

1.21 4.47 0.26 1.16 NR NR

ND. No reaction detected,

NR

native iso-1-cytochrome C in both forms (unmodifiedand methylated PEG preparations). These results suggest that the specificity change is not caused by the modification of the lysine-79 residue. Results from the other variants showed that neither lysine-87 nor lysine-72 are involved in the specificity change (Table 4). All variants also possess Thr102 instead of CyslO2 that is present in the wild-type protein. Replacement of the sole free cysteine sulfhydryl at this position protects the variant against dimerization thereby resulting from intermolecular disulfide bond formation without affecting the catalytic activity.”

hydrogen peroxide and 5.4 times for thianthrene). On the other hand, TMS modification increased the affinity constant for hydrogen peroxide fivefold. This suggested an increase in hydrophobicity of the active-site pocket. Methylester modification does not affect k,,, and KM for thianthrene; nevertheless, a significant increase in the KM value for hydrogen peroxide (hydrophilic substrate) was observed with the Cyt-Met protein. Substrate partition between active site and solvent seems to be an important factor on biocatalysis of hydrophobic substrates by lignin peroxidase and cytochrome C;23 thus, a change in hydrophobicity in the active-site pocket could lead to a change in catalytic behavior. As a consequence of these variations, the catalytic efficiency was ninefold higher for PEG-Cyt than for unmodified protein. The PEG-Cyt-Met preparation showed a four times higher k,,,/K, value than that of native cytochrome. Stability of the chemical modifications of cytochrome C was determined in the presence of I mM hydrogen peroxide. Inactivation constants (K,,) were obtained from a first-order equation (Table 3). All the chemical modifications were less stable than the unmodified horse heart cytochrome. As in the case of site-directed mutagenesis of cytochrome C, a greater catalytic activity is accompanied by a decreased stability of the protein to peroxide.5 Horse heart cytochrome C has a lysine-79 residue which is placed on the window of the solvent-exposed side of heme. Yeast iso-1-cytochrome C also has the lysine 79 in the same position. In order to explore the role of this residue and the other two lysines on the specificity change, we have chemically modified three variants of yeast cytochrome C. As shown in Table 4, no differences in specificity could be found between the K79A and Table 3

Kinetic and inactivation

constants for different chemical Hydrogen

This work has demonstrated that the chemical modification of cytochrome C with poly(eahylene)glycol and methyl alkylation produced a biocatalyst able to oxidize a larger number of PAHs than the unmodified protein. PEG modification increases the catalytic activity of cytochrome C and reduces the KM value for both substrates (hydrogen peroxide and the aromatic compound). Lysine-79, a reactive group placed on the solvent access to the active site, does not seem to be responsible for the specificity change. Thus, chemical modification of biocatalyst seems to be an important tool to design new biocatalyst with environmental proposes. Experimentation is currently being performed in order to determine which reactive group (amino acid residue or functional group) is responsible for this improvement of biocatalytic properties of cytochrome C.

modifications

1.34 1.90 2.13 1.76 0.93

27.4 75.7 3.1 19.0 138.9

of cytochrome

C Thianthrene

peroxide K M.WP (mM)

K M.-VP (rnM)

Preparation Unmodified Cyt Cyt-Met PEG-Cyt PEG-Cyt-Met PEG-Cyt-TMS

Conclusions

48.9 25.1 687.1 92.6 6.7

46.6 41.8 8.5 16.1 47.6

Enzyme Microb.

Technol.,

Kin (mini’) 26.7 45.4 250.6 109.3 19.5

0.035 0.465 0.630 0.239 0.445

1998, vol. 22, January

11

Papers Table 4 Specific activity on a polycyclic aromatic hydrocarbon of iso-I-cytochrome C”

oxidation with unmodified

and modified preparations

of two variants

Specific activity (mini’) Preparation Wild type (WT) K79A K72A K87A PEG-Wl-Met PEG-K79A-Met PEG-K72A-Met PEG-K87A-Met

Pyrene 1.89 1.87 1.25 3.23 12.13 12.02 9.25 11.08

Phenanthrene

(2 0.16) (-’ 0.25) (” 0.12) f ” 0.73) (2 0.25) 1% 0.78) (2 0.87) (k 0.91)

0.90 0.79 1.52 1.05

ILR0.13) (2 0.09) (2 0.01) (2 0.11)

Acknowledgments This work was funded by a DEGAPA-UNAM Grant IN214594 and by the National Council for Science and Technology of Mexico (Grant CONACyT 4217A-9405). Acknowledgment is made to Prof. A. Grant Mauk from the University of British Columbia, Canada for donation of iso- 1-cytochrome C variants. We thank Rosa Roman for technical assistance.

11.

12.

13.

References

10.

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Chrysene

NR NR NR

NR NR NR NR NR NR NR NR

NR NR NR 0.86 0.68 0.23 0.37

No reaction detected, NR a All iso-I-cytochrome variants are double mutants. In addition to the mutation the Cys protecting the variants against dimerization

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(“i”O.12) tt 0.03) (2 0.03) tt 0.09)

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