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Published as doi: 10.1096/fj.05-4173fje.
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(The FASEB Journal. 2006;20:1233-1235.)
© 2006 FASEB

Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering

Brenda Valderrama*,1, Humberto García-Arellano*,2, Stefania Giansanti§, M. Camilla Baratto§, Rebecca Pogni§ and Rafael Vazquez-Duhalt*

* Department of Cellular Engineering and Biocatalysis, Biotechnology Institute, National University of Mexico, AP 510–3, Cuernavaca, México; and

§ Department of Chemistry, University of Siena, Siena, Italy

1Correspondence: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México. AP 510–3, Cuernavaca, Morelos, CP 62250, México. E-mail: brenda{at}ibt.unam.mx

ABSTRACT

Iso-1-cytochrome c, as any other hemeprotein, is able to react with hydrogen peroxide and to engage in the peroxidase cycle. However, peroxidases are irreversibly inactivated by their substrate, hydrogen peroxide. The oxidative inactivation of hemeproteins is mechanism based and arises as the consequence of unproductive electron abstraction reactions. Protein elements, such as the porphyrin ring or the protein backbone, act as simultaneous and competing electron sources even in the presence of exogenous reducing substrates, leading to a decline in activity. It is hypothetically possible to alter the intramolecular electron transfer pathways by direct replacement of low redox potential residues around the active site; as a consequence, the inactivation process would be delayed or even suppressed. To demonstrate this hypothesis, a redox-inspired strategy was implemented until an iso-1-cytochrome c variant fully stable at catalytic concentrations of hydrogen peroxide was obtained. This variant, harboring the N52I,W59F,Y67F,K79A,F82G substitutions, preserved the catalytic performance of the parental protein but achieved a 15-fold higher total-turnover number. The phenotype of this variant was reflected in the stability of its electronic components, allowing identification of a protein-based radical intermediate mechanistically similar to Compound I of classical peroxidases. The results presented here clearly demonstrate that redox-inspired protein engineering is a useful tool for the rational modulation of intramolecular electron transfer networks.—Valderrama, B., García-Arellano, H., Giansanti, S., Baratto, M. C., Pogni, R., Vazquez-Duhalt, R. Oxidative stabilization of iso-1-cytochrome c by redox-inspired protein engineering.


Key Words: hemeproteins • peroxidase • oxidative inactivation • protein engineering

OXIDATIVE HEMEPROTEINS ARE a large class of biocatalysts that function by iron-based oxidation-reduction reaction mechanisms to introduce oxygen into or remove electrons from organic compounds and that participate in many physiological processes, from extracellular lignin degradation to control of oxidative stress. Despite the deep knowledge of their biochemical properties, it is not usually taken into consideration that their functionality might become compromised by their intrinsic instability toward hydrogen peroxide, one of the substrates of the peroxidase cycle that all hemeproteins are potentially able to perform.

In particular, peroxidases are ubiquitous enzymes that catalyze a variety of oxygen transfer reactions using peroxide as the oxidant. The classical reaction catalyzed by peroxidases is oxidative dehydrogenation, although they also catalyze a variety of related reactions, including oxygen transfer, hydrogen peroxide cleavage, and peroxidative halogenations (1) . Although extensively documented, the oxidative sensitivity of classical peroxidases toward hydrogen peroxide has only recently been recognized to be a property shared by all hemeproteins (1) . Further, the oxidative inactivation of hemeproteins seems to be mechanism based, in contrast to the general oxidation of non-hemeproteins by peroxides. Examination of experimental evidence from our groups and from the literature enabled us to formulate a consensus mechanism comprising several stages (1) . The initial radical generation occurs at the heme moiety and gives rise to the formation of protein radicals (1 , 2) . These species are believed to be formed by an initial two-electron oxidation of the iron(III)porphyrin giving rise to Compound I, which consists of a high-valent oxo-iron(IV)porphyrin-based {pi}-free radical cation. In some cases, the second oxidation equivalent in Compound I is delocalized into the protein (3 4 5) . These two oxidizing equivalents are subsequently repaired by two sequential electron transfer reactions to the heme edge. Abundant evidence suggests that in the absence of an exogenous electron donor, enzyme elements such as amino acid residues or the porphyrin moiety itself might perform as potential electron sources (1 , 6 7 8 9) . Nevertheless, and based on its redox potential, Compound I is not expected to oxidize the totality of the natural amino acid residues but only tyrosine, cysteine, tryptophan, and methionine residues (10 , 11) (Fig. 1 A). Oxidation of catalytically important enzyme elements seems to underlie the sensitivity of peroxidases toward its substrate, hydrogen peroxide (1) . Previous experience obtained by the in vitro oxidation of globular proteins revealed that once a protein-based free radical is formed, the fate of free radicals will follow the redox properties of the amino acid side chains and it is expected that they will eventually converge to the lowest redox potential site available (10 11 12) . Transfer of electrons within peptides and proteins appears to be both rapid and efficient, aided by the presence of defined structures such as H-bond networks (10 , 12 , 13) .


Figure 1
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  		Figure 1. A) Oxidation potential of horseradish peroxidase C catalytic intermediates compared with other compounds. B) Amino acid sequence of the iso-1-cytochrome c pseudo wild-type WT16 variant. Oxidation-sensitive residues are highlighted.

In brief, substrate oxidation by hemeproteins seems to be a naturally imperfect process and in the absence of a suitable exogenous reducing substrate the porphyrin ring or the protein backbone may become alternative electron sources. Protein destruction seems to arise as a consequence of unproductive electron abstraction pathways, whereas protection by the reducing substrate comes from the favorable partition of the oxidative equivalents toward the added substrate (1 , 9) . In this paper we present the oxidative stabilization of yeast iso-1-cytochrome c, a model peroxidase (14) , without further compromise of its catalytic properties, through rational alteration of the intramolecular electron transfer networks.

MATERIALS AND METHODS

Site-directed mutagenesis of yeast cyc1
Single and multiple mutants of cyc1 were obtained as described elsewhere by the megaprimer extension method (15) using plasmid pBTR1 as template (16) . All amplifications were performed using high-fidelity polymerase from Altaenzymes (Alberta, Canada) following the manufacturers instructions. Overexpression and purification of mutant proteins were performed as described elsewhere (17) .

Activity assays
Iso-1-cytochrome c reaction mixtures contained 15% acetonitrile, 1.2 µM pynacyanol blue as substrate, and 0.5–3.5 µM of the pure protein preparation in 1 ml of 60 mM sodium phosphate buffer, pH 6.1. Reactions were started by the addition of hydrogen peroxide. Specific activity was determined by measuring the moles of substrate oxidized per mole of iso-1-cytochrome in 1 min and expressed as min–1. All reactions were performed in triplicate. Catalytic parameters were obtained by fitting the experimental data onto the Michaelis-Menten equation using the EnzFitter program from Biosoft (Ferguson, MO, USA).

Stability determinations
All experiments were carried out at room temperature in 60 mM sodium phosphate buffer pH 6.1 with reagents from Sigma (St. Louis, MO, USA), unless otherwise stated. For the inactivation rate calculation, independent iso-1-cytochrome c preparations (1 µM) were incubated in the presence of 1 mM hydrogen peroxide in 0.85 ml buffer for different times until inactivation. The residual peroxidase activity was estimated after the addition of 0.15 ml of 1.2 µM pynacianol blue ({epsilon}603 nm=82.35 mM–1 cm–1) in acetonitrile (American Bioanalytical, Natick MA, USA). Catalytic activity stability values are the activity half-life estimated under these conditions. Soret bleaching was evaluated spectroscopically at 408 nm using a Beckman 650 spectrophotometer (Fullerton, CA, USA). An iso-1-cytochrome c (10 µM) preparation was incubated in the presence of 1 mM hydrogen peroxide in buffer and the absorbance data were recorded from t = 0 every minute until bleached.

Multimerization assays
An iso-1-cytochrome c preparation (10 µM) was incubated in 2 ml buffer in the presence of 1 mM hydrogen peroxide until inactivated. Aliquotes of the mixture (0.1 ml) were removed before the addition of the peroxide and at the end of the incubation and immediately denatured by boiling in one volume of 2x SDS Sample buffer (125 mM Tris-HCl pH 6.8, 1% SDS, 20% glycerol, and 10% ß-mercaptoethanol). All samples were simultaneously separated in a 15% PAGE gel, which was further stained using the non-ammoniacal silver staining method and dried (18) .

Thiodiphenol ionization potential estimation
The ionization potential of thiodiphenol was estimated by the charge transfer method as described elsewhere (19) . Other values were from Aloisi et al. (20) .

Remazol blue turnover number
Samples of pure iso-1-cytochrome c preparations (100–200 nmol) were immobilized onto 0.5 ml of Affi-Gel 15 (Bio-Rad, Hercules, CA, USA) according to the product instructions. The amount of bound protein was estimated after deduction of the nonbound residual protein (quantified with the Bio-Rad protein assay). A mixture of 15 ml of 0.11 mM Remazol brilliant blue R (Reactive blue 19, Sigma) in 60 mM phosphate buffer pH 6.1 was streamed through the packed cytochrome-Affi-Gel preparation. The substrate residual amount was estimated spectroscopically ({epsilon}592 nm=8.48 mM–1 cm–1). Total yield was calculated as the accumulated mole number of substrate molecules utilized by one molar equivalent of iso-1-cytochrome c during the functional life of the enzyme.

Total turnover number estimation
Iso-1-cytochrome c reactions contained 15% acetonitrile, 20 µM substrate, and 0.5–3.5 µM of the pure protein preparation in 1 ml of 60 mM sodium phosphate buffer pH. 6.1. Reactions were started by the addition of 0.5 mM hydrogen peroxide. Samples of 50 µl were taken at different times and the residual amount of substrate was estimated after separation in a Hypersil ODS column (2.1x100 mm, Agilent Technologies, Palo Alto, CA, USA) using a Perkin Elmer 600 series HPLC equipped with a diode array detector. The column was eluted with 30% acetonitrile except for the separation of 4,4'-chloro diphenyl sulfur, where 60% tetrahydrofuran was used. Relative substrate consumption was determined by comparing the area of the remaining substrate at specific wavelengths (4,4'-chloro diphenyl sulfur at 260 nm, 4,4'-thio dianiline at 265 nm, phenyl sulfur, and 4,4'-thio diphenol at 250 nm). Total yield was calculated as the accumulated mole number of substrate molecules used by one molar equivalent of iso-1-cytochrome c during the functional life of the enzyme.

EPR measurements
EPR solutions were prepared with a final concentration of 0.8 mM enzyme and 4 mM hydrogen peroxide (1:5 molar ratio) in 0.1 M phosphate buffer pH = 6.1 The reaction was stopped by rapid immersion of the EPR tube in liquid nitrogen after 10 s and 2 min. CW-X-band (9 GHz) EPR measurements were carried out on a Bruker E500 Elexsys Series using the Bruker ER 4122 SHQE cavity and an Oxford helium continuous flow cryostat (ESR900). The EPR behavior of the untreated WT16 protein and the commercial yeast iso-1-cytochrome c were identical, demonstrating that the T-5A and C102T substitutions were of no relevance (data not shown). Spin quantification was performed by double integration of the experimental EPR radical signal compared with the iron signal.

To assess radical formation under operation conditions, a solution containing 0.5 mM cytochrome c and 2.5 mM hydrogen peroxide in 1 ml 0.1 M phosphate buffer pH 6.1was incubated for 1 min at room temperature, transferred into an EPR tube, and the reaction stopped by rapid immersion into liquid nitrogen. The presence of the radical signal was assessed by direct EPR as described above. A similar mixture but of twice the volume was incubated in the presence of 0.5 mM ABTS for 1 min, one-half of the volume was frozen in liquid nitrogen and used for direct detection of the ABTS radical while the rest of the mixture was passed through a Sephadex G25 size exclusion chromatographic column. The void volume of the column was immediately frozen and used for direct detection of the residual protein-based radical. All the procedure was performed in <40 s.

RESULTS

We have proposed that the actual stability of peroxidases seems to be a consequence of the competition between productive (the added reducing substrate) and unproductive electron sources (catalytically important enzyme components) (1 , 9) . This assumption suggests that the redox properties of the added substrate would influence the electron-abstraction partition between sources controlling the stability of the enzyme under normal operation conditions. To demonstrate this, we calculated the total turnover number of an iso-1-cytochrome c preparation using members from a set of diphenyl sulfur derivatives with different ionization potential values as substrates (Fig. 2 ). We found a good correlation between the substrate ionization potential value and the total turnover number of the preparation with a ~30-fold extension of the operational life of the enzyme. The evidence that the oxidative stability of iso-1-cytochrome c as a peroxidase can be enhanced by including exogenous substrates with lower redox potential supports the existence of simultaneous and competing electron sources.


Figure 2
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  		Figure 2. Relationship between the ionization potential of a series of substituted derivatives of diphenyl sulfide and the total turnover number of an immobilized yeast iso-1-cytochrome c preparation (moles of oxidized substrate per mole of immobilized enzyme until inactivation).

Our experimental strategy was then aimed at enhancing the partition of the oxidizing equivalents toward the exogenous substrate instead of the protein elements. To do this, we decided to selectively replace unproductive electron sources and, concomitantly, to alter the intramolecular electron transfer pathways. Among the redox-sensitive amino acid side chains shown in Fig. 1A , Tyr residues present the lowest redox potential and emerged as the most probable final electron donor. Yeast iso-1-cytochrome c harbors five Tyr residues at positions 46, 48, 67, 74, and 97 (Fig. 1B ). The involvement of Tyr residues during iso-1-cytochrome c inactivation was confirmed by the identification of dityrosine-based oligomers (Fig. 3 ). Similar oligomers have been observed during the oxidative inactivation of other hemeproteins, suggesting a common mechanism (1 , 21) . To test the consequence of removing these final electron sources, we constructed an iso-1-cytochrome c variant where all five Tyr residues were substituted for Phe side chains, which resulted in oligomerization abolition (Fig. 3) . This variant preserved 77% of the activity, enhanced the Soret band half-life by 2.6-fold and the affinity for hydrogen peroxide by 12-fold, but decreased the stability of the preparation to only 8.3% (Table 1 ). Whereas the enhanced affinity for hydrogen peroxide might be due to the widening of the heme crevice, the improved porphyrin stability and the drastic instability of the activity can be further explained as the consequence of a novel organization of the electron transfer pathways, reduced from the porphyrin toward the heme iron but enhanced from the protein. Of the five mutated residues, the OH group from residues 74 and 97 points toward the outskirts of the protein; although they might be involved in the oligomerization mechanism, they do not interact with the porphyrin. In contrast, the OH groups from Tyr 46, 48, and 67 interact with the heme group in a complex H-bond network, connecting the heme iron with the protein backbone through the porphyrin (22) .


Figure 3
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  		Figure 3. Denaturing PAGE of yeast iso-1-cytochrome c variants before and after extensive incubation with 1 mM H2O2. Lanes 1 and 5, MW markers. Lane 2, untreated WT16 variant. Lane 3, H2O2-treated WT16 variant. Lane 4, H2O2-treated Y46F,Y48F,Y67F,Y74F,Y97F variant.


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  		Table 1. Stabilities of activity and heme integrity in the presence of 1 mM hydrogen peroxide and catalytic constants of different yeast iso-1-cytochrome c variants

While there is no information available regarding the potential role of residues 46 and 48 in the electron transfer properties of iso-1-cytochrome c, Y67 performs a dual regulatory task: it sets the midpoint reduction potential through interaction with the sulfur atom of the M80 ligand and stabilizes the alternative oxidation states of the protein as an electron carrier (23) . The regulatory function of Y67 is achieved through a well-delineated and flexible hydrogen bond network comprising the porphyrin moiety, the conserved water molecule Wat166, and other amino acid residues such as N52 (24) . The crystallographic structure of an iso-1-cytochrome c N52I,Y67F double mutant has been published (25) . The most prominent difference observed in this mutant relative to the wild-type is the displacement of Wat166, a molecule that performs multiple structural and functional roles through its associated H-bond network (25 , 26) . In consequence, the internal H-bond network was drastically altered in the double mutant and may provide an appropriate scenario for evaluating the consequence of isolating the porphyrin ring from the protein background. We decided then to analyze the catalytic properties of a previously constructed N52I,Y67F double mutant (27) .

Disruption of the Wat166-related H-bond network in the double mutant abated its peroxidase activity below 0.1% enhanced the affinity for hydrogen peroxide by 25-fold, but decreased the stability of the enzyme to only 6.1% compared with WT16 (Table 1) . In agreement with previous observations, stabilization of the Soret band was absolute and no bleaching could be observed even after several hours exposure to 1 mM H2O2 (27) . The higher affinity for hydrogen peroxide is comparable to that observed in the tyrosine-less mutant and can be explained similarly. In contrast, the reduced catalytic activity and the evident heme group stability suggest that the added substrate and the porphyrin are no longer used as final electron donors, but only the protein scaffold, which might explain the reduced stability of the enzyme. At this point, we are able to draw two significant conclusions. First, that the hypothesis of simultaneous and competing use of different protein elements as electron sources seems valid, since we have clear evidence of dissociation. Second, the dissociation profile can be modulated by the rational remodel of the intramolecular electron transfer pathways.

The fundamental principle underlying our approach to the oxidative inactivation phenomenon is that it is mechanism based (1) . That is, in the absence of a functional catalytic cycle, no oxidative damage should be expected. In particular, the poor catalytic performance of the N52I,Y67F double mutant raises the question of whether the apparent porphyrin stability could be due to its low turnover number and not to a distorted use of alternative electron sources. The only solution to this subject was to further modify the mutant iso-1-cytochrome c internal cavity in order to restore the catalytic performance of the wild-type.

Aside from tyrosine, there are other oxidation-sensitive residues in iso-1-cytochrome c such as Cys, Met, and Trp (Fig. 1A ). Although wild-type yeast iso-1-cytochrome c contains three Cys residues at positions 14, 17, and 102, our pseudo wild-type WT16 variant lacks C102 as a means to evade the natural dimerization of the protein (Fig. 1B ). The sulfhydryl groups of residues 14 and 17 participate in the covalent bonding of the porphyrin as thioether linkages, enhancing the redox potential of the sulfur atoms and significantly reducing their oxidative sensitivity. According to the translated sequence of wild-type iso-1-cytochrome c, there are three Met residues at positions 1, 64, and 80 (Fig. 1B ). M1 is not observed in the crystallographic model of the protein (22) either because of its high thermal mobility or because it was actually removed during protein maturation, in any case it is not likely to contact the porphyrin ring. While residues 64 and 80 are in close contact with the heme group, the side chain of the variable M64 is not bonded to any other protein element but participates in the conformation of the hydrophobic environment inside the internal cavity. In contrast, the sulfur atom from M80 is intimately bound to the heme group as the sixth ligand of the iron atom (28) . M80 is an invariant residue with a central role in the complexation interactions formed with electron transfer partners and its interaction modulates the heme reduction potential (28) .

The relevance of M80 in the redox properties of iso-1-cytochrome c prompted our interest in elucidating its possible role during the oxidative inactivation of the protein. To achieve this, we constructed and analyzed a N52I,Y67F,M80A triple mutant (Table 1) . Disruption of the sixth iron ligand returned the activity half-life and the affinity for hydrogen peroxide to levels similar to those observed in WT16, and resulted in a modest enhancement of the peroxidase activity still below 2% of the WT16 values. In contrast, the stability of the heme group was severely compromised, probably due to the turnover number enhancement. These results suggest that while the contribution of the axial sulfur-iron bond to the oxidative inactivation of the N52I,Y67F double mutant is marginal, other oxidation-sensitive residues could be involved.

The iso-1-cytochrome c from yeast presents a single Trp residue at position 59 (Fig. 1B ) (26) . The N1H atom of the indole ring is H-bonded to the rear heme propionate and, based on the conserved nature of the residue, this bond is likely to be important to the structural and functional properties of cytochromes c. Two roles have been proposed for W59. One is that it helps to provide a stabilizing hydrophobic core to the protein by its large aromatic side chain (29 , 30) . The second, and more relevant role for this work, is that the indole nitrogen helps to modulate the electron transfer properties between the heme group and the protein (31 , 32) .

To trim the H-bond network connecting the heme propionates with the protein backbone even further, we performed a site-directed substitution of the single Trp residue for Phe in the N52I,Y67F background (Table 1) . While the activity and heme stabilities were not significantly altered, the specific W59F substitution improved the catalytic activity of the double mutant by ~900-fold, up to levels similar to those observed in WT16. Significant restoration of the added substrate utilization while preserving the Soret band integrity indicates that the porphyrin was actually stabilized.

Furthermore, these results suggest that we kept the radical transfer path from the porphyrin toward the iron atom interrupted while restoring the efficient utilization of added substrates as electron donors. Nevertheless, the stability of the catalytic activity was not improved, suggesting that other protein components were still being damaged. As mentioned, for a free radical to be transferred in a protein both the receptor and the donor must be bonded (12) . That means that in the N52I,W59F,Y67F triple mutant a residual interaction must still bind the porphyrin with the rest of the protein.

According to the 3-dimensional structure of yeast iso-1-cytochrome c, F82 is located in the distal side of the heme where it shapes part of the heme binding pocket (22) . The phenyl ring of this residue is adjacent to pyrrole rings B and C (within ~5 Å) and is almost coplanar with them, leading to the proposal that the delocalized {pi}-electron systems of these groups are coupled, a property not shared by any other residue in the heme pocket (33) . This property coincides with the proposed role of F82 as provider of an optimal medium along the electron transfer path toward the physiological acceptor (34 35 36) . For this reason, we considered that F82, though not apparently bonded, may constitute the residual link between the heme and the protein backbone.

The specific substitution of F82 for Gly in the N52I,W59F,Y67F triple mutant background resulted in the dramatic stabilization of the peroxidase activity, while the porphyrin integrity and the catalytic activity of the parental variant were kept untouched (Table 1) . The activity half-life of the N52I,W59F,Y67F,F82G variant was increased ~10-fold compared with the WT16 protein and ~200-fold compared with the parental variant in the presence of 1 mM hydrogen peroxide.

Despite its substantial stabilization and higher affinity for hydrogen peroxide, the catalytic activity of the N52I,W59F,Y67F,F82G mutant variant was still below 75% of the WT16 value. This behavior could be explained by a reduced access of the substrate to the porphyrin moiety, probably caused by the accumulated cavity reorganization mainly due to the F82G mutation. The crystal structure of yeast iso-1-cytochrome c shows the heme crevice as partially occluded by a H-bond interaction between the side chains of K79 and S47 (22) . In principle, disruption of this interaction may have a positive effect in the peroxidase activity of the protein by allowing a faster exchange rate between the active site and the surrounding medium. In a preliminary experiment, site-directed substitution of this residue for Ala in the WT16 background resulted in a ~7-fold increase of the catalytic activity of the enzyme with no stabilization consequences (data not shown).

As can be seen in Table 1 , not only the catalytic performance of the N52I,W59F,Y67F,K79A,F82G variant improved to ~90% of the WT16 value, but also the stability of the enzyme reflected the consequences of the substitution. Furthermore, the peroxidase activity was completely stabilized and no inactivation could be observed after 4 h incubation in the presence of 1 mM hydrogen peroxide. The affinity for hydrogen peroxide was slightly reduced compared with the parental variant, but the catalytic efficiency of the N52I,W59F,Y67F,K79A,F82G variant was restored to ~85% of the WT16 value.

At this point it might be considered that the stabilization of yeast iso-1-cytochrome c as a peroxidase by our method was only apparent, since our assay conditions did not include an exogenous electron donor. To demonstrate the actual stability of our variants, we estimated the total turnover number of some of them after immobilization in Affi-Gel-15 using a water soluble substrate, Remazol blue (Table 2 ). Despite the low turnover numbers, typical of isocytochrome c as a peroxidase, the yield of the preparations reflected the stability values obtained in the absence of added substrate with a significant improvement (15-fold) compared with WT16.


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  		Table 2. Total turnover number of immobilized wild-type and mutant iso-1-cytochrome c variants under operation conditionsa

As mentioned above, the peroxidase reaction mechanism involves the formation of free radicals, in the porphyrin and also in the protein (37) . To further characterize the electronic properties of the N52I,W59F,Y67F,F82G and N52I,W59F,Y67F,K79A,F82G variants, we performed the direct EPR spectra of these mutants and of WT16 as described in the methods section (Fig. 4 ). We classified the paramagnetic signals observed in the EPR spectrum of WT16 as belonging to different groups based on accepted criteria (38 , 39) : 1) low-spin Iron(III)Porphyrin species identified by the g1 = 3.05 and g2 = 2.33; 2) high-spin Iron(III)Porphyrin species identified by the g{perp} = 6.0 and g|| = 2.0; 3) Iron(III) identified by the g = 4.3 broad and slightly rhombic resonance; and 4) an intense radical-like signal centered at g = 2.004 ± 0.0005. That this signal comes from a protein-based radical was supported by experiments performed at different temperatures and microwave powers (data not shown).


Figure 4
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  		Figure 4. X-band EPR spectra of yeast iso-1-cytochrome c variants after the incubation with 4 mM hydrogen peroxide for different times and rapid freezing in liquid nitrogen. A) WT16 variant. B) N52I,W59F,Y67F,F82G mutant. C) N52I,W59F,Y67F,K79A,F82G mutant. D) Y46F,Y48F,Y67F,Y74F,Y97F mutant. All spectra were recorded at {nu} = 9.390 GHz, 10 G modulation amplitude, 2 mW microwave power and 100 kHz modulation frequency.

The combined porphyrin-bound high- and low-spin iron species represent the population of iron atoms present in the active site. The distribution depends apparently on the strength of the sixth coordination bonding by Met80. In WT16, ~80% of the Iron(III)porphyrin species were in a low-spin configuration (Fig. 4a ). After the addition of hydrogen peroxide to WT16, all the porphyrin-bound iron signals decreased with time and completely disappeared after 2 min incubation. In contrast, the protein-bound signal with g value of 2.004 ± 0.0005 appeared after 10 s and increased with time. The yield of this radical species was 20% after 10 s and 46% after 2 min. The intensity of the g = 4.3 signal remained essentially unaltered and corresponds to a small amount of rhombic nonheme iron impurity often seen in protein samples.

In the stable N52I,W59F,Y67F,F82G variant, the Iron(III)porphyrin high- and low-spin signals were clearly defined and corresponded to the g values observed in WT16 (Fig. 4b ). The broad and slightly rhombic g = 4.3 signal was also present. The relative intensity of the g{perp} = 6.0 and the g1 = 3.05 signals indicated that > 90% of the porphyrin-bound iron was in the low-spin configuration, suggesting a strong axial bonding by Met80. The addition of hydrogen peroxide to this variant had no effect on the iron-related signals. The protein-bound g = 2.004 ± 0.0005 radical was formed after the addition of hydrogen peroxide but was less intense than in WT16.

The other iso-1-cytochrome c variant studied (N52I,W59F,Y67F,K79A,F82G) was a derivative of N52I,W59F,Y67F,F82G with higher catalytic activity due apparently to the opening of the heme crevice. The resting EPR spectrum of this mutant was similar to the one from the N52I,W59F,Y67F,F82G variant, with ~93% of the porphyrin-bound iron in low-spin configuration (Fig. 4c ). The addition of hydrogen peroxide had no deleterious effect on the Iron(III)porphyrin species, with a slight increase of the low-spin signals intensity. There was a 2-fold enhancement of the g = 4.3 signal but the shape of the resonance remained broad and slightly rhombic, with an evident shoulder at the low-field side of the resonance. The protein-bound g = 2.004 ± 0.0005 radical was formed after the addition of hydrogen peroxide and was more intense than in the N52I,W59F,Y67F,F82G mutant but not as intense as in WT16.

In all cases, the protein-bound radical signal presented the same g value (g=2.004±0.0005). The presence of a porphyrinyl radical may be ruled out given the absence of the characteristic broad band signal extending over 2500 G with a g|| = 2.40 at pH = 7.7 and g|| = 2.67 at pH = 4.5 (40) , it therefore being possible for this radical to be based on an amino acid residue. According to the experimental data presented above (Fig. 3) , a probable site for the g = 2.004 ± 0.0005 protein-based radical would be a tyrosine residue. To support this hypothesis, we performed EPR analysis of the Y46F, Y48F, Y67F, Y74F, Y97F variant (Fig. 4d ). In the resting spectrum, the Iron(III)porphyrin high-spin signals at g{perp} = 6.0 and g|| = 2.0 were clearly defined, as well as the g = 4.3 rhombic signal. In contrast, the Iron(III)porphyrin low-spin signals at g1 = 3.05 and g2 = 2.33 were heavily distorted, including a shoulder on the low-field side of the g1 = 3.05 resonance. The addition of hydrogen peroxide resulted in the accumulation of a new species with g|| = 2.52 (band around 2500G) and g{perp} = 2.0 values. This species is probably evidence of the formation of a Compound I intermediate harboring a porphyrin-based radical. The trend of the intensity of the radical signal at g ~ 2.00 with respect to that in the case of WT16, supports this assignment. Additionally, both the concentration and shape of the g = 4.3 resonance were significantly affected in response to the addition of hydrogen peroxide, acquiring high intensity and a more evident rhombic character, which is indicative of chemical modification in the protein.

The fact that in the stable variants the addition of hydrogen peroxide still elicits the appearance of the g = 2.004 ± 0.0005 protein-based radical raises reasonable doubt about the role of this species as an inactivation intermediate. To demonstrate the accumulation of this radical species under noninactivating conditions, we performed the EPR spectra of the most stable variant, N52I,W59F,Y67F,K79A,F82G, in the presence of ABTS as the exogenous reducing substrate (Fig. 5 ). Previous experiments showed that this variant is stable even in the absence of an exogenous substrate; nevertheless, the reaction was stopped after only 2 min by removal of both ABTS and ABTS+• by size exclusion chromatography. The unequivocal identification of a residual signal with similar magnitude and g value as in the control experiment, performed under the same conditions but in the absence of ABTS, strongly suggests that this radical species might be involved in catalysis. The same experiment was performed with commercial iso-1-cytochrome c and with WT16, yielding equivalent results (data not shown).


Figure 5
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  		Figure 5. Assessment of the protein-based g = 2.004 ± 0.0005 radical species as a catalytic intermediate. a) Control spectrum of the N52I,W59F,Y67F,K79A,F82G A variant after 2 min. incubation with hydrogen peroxide. b) Same experiment as in panel a but in the presence of 0.5 mM ABTS. Only the intense ABTS+• signal is detected. c) Spectrum of the mixture analyzed in panel b after removal of ABTS+• by size exclusion chromatography.

DISCUSSION

We previously described the oxidative inactivation of hemeproteins as an unusual case of enzymatic performance, where the enzyme itself is used as electron source and therefore should be considered in the stoichiometry of the reaction (1 , 9) . We assumed the inactivation to be a consequence of multiple simultaneous oxidations susceptible to be observed and altered independently. Based on these premises, here we proposed and developed a protein engineering strategy aimed at remodeling critical electron transfer contacts. This was accomplished by the selective disruption of redox-sensitive residues without further compromise of the activity, resulting in a variant with enhanced stability.

The mechanism-based oxidative inactivation of hemeperoxidases has been addressed from different standpoints, including site-directed mutagenesis and directed evolution (41 42 43 44) . In some cases oxidative stability was achieved as a consequence of reduced activity. In others, since the mutant protein was not purified and characterized, it is difficult to dissociate higher accumulation from actual stabilization. In general, and despite continuous efforts, directed evolution strategies had not been successful in the isolation of more stable peroxidases.

A possible explanation for this shortfall of supportive results arises from the study of the evolution of physiological electron tunneling in oxidoreductases (45 , 46) . Contrary to theoretical predictions, the view that the amino acid matrix has been optimized naturally to guide electron tunneling seems to lack experimental support. In contrast, the single characteristic that has been selected in the evolution of electron tunneling in oxidoreductases is the simple positioning of redox centers within reach. This position considers that almost every change of an amino acid will provide alternative bonds to form a comparable pathway, including water molecules. Even further, conserved amino acids and structural motives in these regions may have many other biologically important functions with their own selective pressures not directly related to electron tunneling.

After these considerations, we support the view that most of the primary sequence of the protein is irrelevant for the electron transfer, since the chemical properties of the residues involved in the pathways are less critical that the residue packing between redox centers (45). As a consequence, only a redox-inspired strategy is in principle likely to succeed in improving a complex circuitry such as the intramolecular electron transfer during the peroxidases inactivation, as demonstrated in this article.

It has been concluded that the generation of amino acid-based radicals in iso-1-cytochrome c after exposure to hydrogen peroxide occurs by a peroxidase-type mechanism, although none of the catalytic intermediates have been characterized to date (47) . Classical Compound I consists in a high-valent oxo-iron(IV)porphyrin-based {pi}-free radical (1) . In some cases, the second oxidation equivalent in Compound I is delocalized into the protein (3 4 5) . In this regard, the identification of a protein-based radical with g value of 2.004 ± 0.0005 in WT16 can be compared with the formation of amino acid-based free radicals in other hemeproteins (1 , 6 , 8 , 21) . This signal appeared in WT16 after 10 s exposure to hydrogen peroxide (1:5, protein:H2O2) and increased even further after 2 min incubation probably due to the progressive delocalization of the electron in the protein with the concomitant decrease in the iron signal (Fig. 4a-c ). Taking into consideration that this protein-based radical is present in the stable variants and that it accumulated even under noninactivating conditions (Fig. 5) , we suggest that in iso-1-cytochrome c, this radical is probably a catalytic intermediate, mechanistically equivalent to the secondary species of Compound I. Furthermore, the site of this radical seems to be a tyrosine residue. The selective substitution of all tyrosine positions for phenylalanine evidenced the transient formation of a classical Compound I intermediate harboring a porphyrin-based radical (g||=2.52 and g{perp}=2.0 values). After 2 min incubation, the g{perp} = 2.0 signal decreased as a very intense signal with g = 4.3 value accumulated. (Fig. 4d ). Since the EPR bands reflecting the porphyrin-bound iron status were not drastically affected, we suggest that in the absence of tyrosine residues, an initiating radical would be transferred around the protein until reaching the porphyrin as a secondary site; nevertheless, this secondary site is not stable and eventually the electron determines some modification at the expense of iron.

In conclusion, the rational strategy applied on iso-1-cytochrome c yielded a more stable protein toward the oxidative inactivation by hydrogen peroxide. The phenotype of this variant was reflected in its electronic properties, allowing identification of a tyrosine residue as the site of a protein-based radical intermediate mechanistically similar to Compound I. Since the design relied on the oxidation sensitivity of the enzyme components more than in the structure, this strategy might be applied to any hemeprotein given the sequence is known.

ACKNOWLEDGMENTS

We thank Prof. Riccardo Basosi for making the EPR facilities available and Dr. Jan Schwarzbauer for his generous gift of 4,4'-dichlorodiphenylsulfide. We thank Raunel Tinoco and Rosa Román for their technical assistance and Prof. Agustín López-Munguía, Prof. Michael A. Pickard and Dr. Jorge Verdín for their valuable scientific advice. This study was supported by DGAPA Grant IN202305–2, IFS Grant F/3562–1, and Conacyt-Semarnat Grant 2004-C01–272.

FOOTNOTES

2 Present address: Department of Molecular Biology and Biotechnology, Biomedical Research Institute, National University of Mexico, AP 70228, México, D.F., México.

Received for publication May 12, 2005. Accepted for publication February 14, 2006.

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