A Cytochrome C Variant Resistant to Heme Degradation by Hydrogen Peroxide Jose a Villegas1, a Grant Mauk2 and Rafael Vazquez-Duhalt1
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cm7401.qxd 03/22/2000 08:20 Page 237 Research Paper 237 A cytochrome c variant resistant to heme degradation by hydrogen peroxide Jose A Villegas1, A Grant Mauk2 and Rafael Vazquez-Duhalt1 Background: Cytochrome c has peroxidase-like activity and can catalyze the Addresses: 1Instituto de Biotecnologia UNAM, oxidation of a variety of organic substrates, including aromatic, organosulfur and Apartado Postal 510-3, Cuernavaca, Morelos, 62250 México. 2Department of Biochemistry and lipid compounds. Like peroxidases, cytochrome c is inactivated by hydrogen Molecular Biology, University of British Columbia, peroxide. During this inactivation the heme prosthetic group is destroyed. Vancouver, British Columbia, Canada V6T 1Z3. Results: Variants of the iso-1-cytochrome c were constructed by site-directed Correspondence: Rafael Vazquez-Duhalt mutagenesis and were found to be more stable in the presence of hydrogen E-mail: [email protected] peroxide than the wild type. No heme destruction was detected in a triple Key words: biocatalysis, cytochrome c, inactivation variant (Tyr67→Phe/Asn52→Ile/Cys102→Thr) with the catalytic hydrogen mechanism, peroxidase activity, stability peroxide concentration of 1 mM, even following the loss of catalytic activity, whereas both double variants Tyr67→Phe/Cys102→Thr and Asn52→Ile/ Received: 18 November 1999 → Revisions requested: 15 December 1999 Cys102 Thr showed a greater rate of peroxide-induced heme destruction than Revisions received: 4 January 2000 observed with the wild-type protein. Accepted: 13 January 2000 Conclusions: Heme destruction and catalytic inactivation are two independent Published: 7 March 2000 processes. An internal water molecule (Wat166) is shown to be important in Chemistry & Biology 2000, 7:237–244 the heme destruction process. The absence of a protein radical in the resistant variant suggests that the protein radical is necessary in the heme destruction 1074-5521/00/$ – see front matter process, but presumably is not involved in the reactions leading up to the © 2000 Elsevier Science Ltd. All rights reserved. protein inactivation. Introduction the absence of reducing substrate [8,15]. The substrate Cytochromes c, which are involved in the electron trans- inactivation of peroxidases leads to the modification of the port system, can catalyze peroxidase-like reactions in vitro heme prosthetic group and probably to the formation of a [1]. As observed for peroxidases, the heme prosthetic verdohemoprotein as a final product [16]. The inactivation group of cytochrome c is activated by an electron accep- mechanism of peroxidases, however, has not yet been tor, such as hydrogen peroxide or an organic hydroperox- clearly elucidated. Recently we have reported that vari- ide. No catalytic activity of cytochrome c has been ants of iso-1-cytochrome c have differing stability in the described in living systems. More than 45 years ago, presence of hydrogen peroxide when compared with the however, the ability of cytochrome c to induce lipid per- wild-type protein [1,10]. We are currently using site- oxidation, as well as to promote hydroperoxide cleavage, directed mutagenesis to study the inactivation mechanism was reported [2]. Cytochrome c has subsequently been of cytochrome c and for the rational design of a new and reported to catalyze the hydroxylation of 4-nitrophenol more stable biocatalyst. In this study, we report a triple [3], the oxidation of 2-keto-4-thiomethyl butyric acid [4], variant in which the heme prosthetic group is not the oxidation of (2-2′-azino-bis(3-ethylbenzthiazoline-6- degraded by and catalytic concentrations of hydrogen per- sulfonic acid (ABTS) diammonium salt), and the oxida- oxide. The differentiation of the inactivation and the tion of 4-aminoantipyrine [5]. Cytochrome-P450-like heme destruction processes in cytochrome c exposed to oxidative reactions with free and immobilized cytochrome peroxide are discussed. c (e.g., N- and O-demethylation, S-oxidation and epoxida- tion of olefins) are performed in the presence of hydrogen Results peroxide or organic hydroperoxides [6]. These activities During catalytic turnover, iso-1-cytochrome c is inacti- have led to the use of cytochrome c as a biocatalyst in the vated by hydrogen peroxide. The stability of cytochrome c oxidation of thiophenes and organosulfides to form sulfox- was determined by incubating the protein with 1 mM ides [7,8] and aromatic compounds [9,10]. H2O2, which is the concentration of peroxide used in the biocatalytic activity assay (see the Materials and methods As with horseradish peroxidase [11], lignin peroxidase section). After 10 minutes, the cytochrome lost 75% of its [14] [12], manganese peroxidase [13], lactoperoxidase , and activity (Figure 1), with an inactivation rate constant (kin) other peroxidases, cytochrome c is inactivated by an of 0.132 min–1 determined from fitting the data to a first- excess of hydrogen peroxide or organic hydroperoxides in order kinetic model. Comparing the inactivation constants cm7401.qxd 03/22/2000 08:20 Page 238 238 Chemistry & Biology 2000, Vol 7 No 4 Figure 1 Figure 2 The effect of hydrogen peroxide concentration on the rate constant for inactivation of iso-1-cytochrome c. during inactivation. Some changes in the amino acid com- Inactivation and heme destruction of iso-1-cytochrome c in the position were found after incubation (Figure 4). All five presence of 1 mM H2O2. tyrosine residues and three cysteine residues disappeared after the 8 minute treatment. The amino acid composition of untreated protein, however, showed that only four at 1 mM H2O2 showed that yeast cytochrome c is less tyrosines and only one cysteine could be detected because stable than peroxidases such as chloroperoxidase from Cal- the other two cysteines are covalently bonded to heme. In –1 dariomyces fumago (kin = 0.006 min ), horseradish peroxi- addition, one methionine and one lysine appear to be –1 –1 dase (kin = 0.021 min ), lactoperoxidase (kin = 0.034 min ) transformed. No significant changes could be detected in –1 and lignin peroxidase (kin = 0.111 min ), and manganese the amounts of the other amino acids following inactiva- –1 peroxidase (kin = 0.082 min ) from Phanerochaete chrysospo- tion of the cytochrome with hydrogen peroxide. rium. The cytochrome c inactivation constant increases with increasing hydrogen peroxide concentration, as is From these results, it appears that modification of the observed for the peroxidases (Figure 2). protein structure could be the origin of the inactivation. In the presence of hydrogen peroxide, heme proteins are During the inactivation process, the heme prosthetic known to form protein radicals located primarily on tyro- group is degraded, as indicated by the diminished sine residues [17]. Tyrosine radical formation is involved in absorbance in the Soret region (Figure 3). Both heme the peroxide-based dimerization [18,19] of myoglobin and destruction and the loss of catalytic activity exhibit first- in the formation of a covalent bond between apo-myoglo- order kinetics, but the rate constants for the two processes bin and the heme prosthetic group [20]. For horse heart differ (Figure 1). After eight minutes of incubation with cytochrome c, it has been suggested that initial oxidation of hydrogen peroxide, the cytochrome lost 64% of its activ- the heme by hydrogen peroxide results in a ferryl ity, whereas only 30% of the heme has been destroyed, (FeIV = O) species. The radical might then undergo an according the Soret band (Figure 1). The inactivation intramolecular transfer to a tyrosyl residue [21]. To explore process therefore appears to precede the heme destruction the involvement of tyrosine free radicals following reaction process. To corroborate the heme destruction results, the of cytochrome c with peroxide, a variant of iso-1-cyto- rate of iron release was measured using atomic absorption chrome c devoid of tyrosine (Y–) was prepared using the spectroscopy. After 10 minutes incubation in the presence polymerase chain reaction (PCR) with various amplifica- of 1 mM H2O2, 63% of iron present in the protein is tions steps. After purification, the stability of the multiple released and can be detected as protein-free iron in the variant in the presence of 1 mM H2O2 was assayed, and a –1 supernatant solution, after protein precipitation. No kin of the variant was 0.1139 min — a value very similar to –1 soluble iron was detected in control samples incubated that observed for the wild-type protein (kin = 0.132 min ). without hydrogen peroxide and extracted in the same Because our main interest in cytochrome biocatalysis is manner as the experimental samples. polycyclic aromatic hydrocarbon oxidation [1,10], we have tested the different variants for pyrene oxidation. The Amino acid analyses were carried out before and after variant that did not contain tyrosine also showed reduced 8 minutes incubation with hydrogen peroxide to deter- specific activity for pyrene oxidation (1.45 min–1), relative mine whether proteic moiety also undergoes modification to that of the wild-type protein (2.8 min–1). cm7401.qxd 03/22/2000 08:20 Page 239 Research Paper Cytochrome c variant resistant to hydrogen peroxide Vazquez-Duhalt et al. 239 Figure 3 Eight yeast cytochrome variants were assayed for stability at varying hydrogen peroxide concentrations (Table 1). The double variants Phe82→Ala/Cys102→Thr (F82A/ C102T), Lys72→Ala/Cys102→Thr (K72A/C102T), and Lys79→Ala/Cys102→Thr (K79A/C102T) were less stable than the wild-type cytochrome in the presence of 1 mM → → H2O2, whereas the Lys73 Ala/Cys102 Thr (K73A/ C102T), Lys87→Ala/Cys102→Thr (K87A/C102T) and Asn52→Ile/C102T (N52I/C102T) variants were signifi- cantly more stable. In the case of the Tyr67→Phe/C102T (Y67F/C102T) variant, stability in the presence of 1 mM H2O2 is similar to that of the wild-type protein. Neverthe- less, this variant is less sensitive to increased hydrogen peroxide concentration (Table 1). All these variants were catalytically active, and they showed specific activities for the pyrene oxidation of 1.3–3.3 min–1, whereas the spe- cific activity of the wild-type cytochrome was of 2.8 min–1.