Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Peroxidase and Photoprotective Activities of Magnesium Protoporphyrin IX

  • Kim, Eui-Jin (Department of Life Science and Basic Science Institute for Cell Damage Control, Sogang University) ;
  • Oh, Eun-Kyoung (Department of Life Science and Basic Science Institute for Cell Damage Control, Sogang University) ;
  • Lee, Jeong K. (Department of Life Science and Basic Science Institute for Cell Damage Control, Sogang University)
  • Received : 2013.11.26
  • Accepted : 2013.12.05
  • Published : 2014.01.28
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Abstract

Magnesium-protoporphyrin IX (Mg-PPn), which is formed through chelation of protoporphyrin IX (PPn) with Mg ion by Mg chelatase, is the first intermediate for the (bacterio)chlorophyll biosynthetic pathway. Interestingly, Mg-PPn provides peroxidase activity (approximately $4{\times}10^{-2}units/{\mu}M$) detoxifying $H_2O_2$ in the presence of electron donor(s). The peroxidase activity was not detected unless PPn was chelated with Mg ion. Mg-PPn was found freely diffusible through the membrane of Escherichia coli and Vibrio vulnificus, protecting the cells from $H_2O_2$. Furthermore, unlike photosensitizers such as tetracycline and PPn, Mg-PPn did not show any phototoxicity, but rather it protected cell from ultraviolet (UV)-A-induced stress. Thus, the exogenous Mg-PPn could be used as an antioxidant and a UV block to protect cells from $H_2O_2$ stress and UV-induced damage.

Keywords

Introduction

Animals, plants, and aerobic microorganisms respire to acquire ATP through the electron-transfer chain in the membrane, using O2 as a terminal electron acceptor. Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radical are often generated owing to the incomplete reduction of O2. DNA mutation, protein denaturation, and lipid peroxidation could be observed through the ROS-induced oxidation [2,29]. Thus, antioxidant enzymes are present in most organisms to overcome the oxidative stress, and include superoxide dismutase (SOD), catalase, and peroxidase [14]. Although both catalase and peroxidase have the ability to detoxify H2O2, their mechanism to remove H2O2 is different from each other [1,5,34]. Catalase mediates the redox reactions with H2O2, generating H2O and O2 as the reaction products [1], whereas peroxidase reduces organic peroxide (ROOH: if R is H, it is H2O2) to form ROH and H2O with the concomitant oxidation of the electron donor [5,34].

The metabolic pathway from δ-aminolevulinic acid (ALA) to PPn is shared by the biosynthesis of both heme and (bacterio)chlorophyll [22,40]. In animals, fungi, and a few bacteria such as purple nonsulfur photosynthetic bacteria, ALA is synthesized through decarboxylative condensation of glycine and succinyl-CoA [42]. In plants, algae, and most bacteria, ALA is alternatively synthesized from the reduction of glutamyl-tRNA, followed by aminomutase reaction [4]. Ferrochelatase [8]mediates the coordination of Fe2+ within PPn to form Fe-PPn, also known as heme b or protoheme IX, whereas Mg chelatase mediates the coordination of Mg2+ within PPn to form Mg-PPn, the first committed precursor for the biosynthesis of (bacterio)chlorophyll [6,24].

Antioxidative activities of metalloporphyrins to detoxify superoxide and H2O2 have been reported [10,27,35]. Manganese (Ⅲ) meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP) and Mn (Ⅲ) meso-tetrakis (1-methyl-4-pyridyl) porphyrin (MnMPyP) showed abilities to remove superoxide as well as H2O2 [10]. When Mn ions at the center of MnTBAP and MnMPyP are replaced by zinc ion (Zn), catalase-like activity, though significantly reduced in comparison with that of the Mn-containing porphyrins, is still observed [10]. In addition, it was reported that the H2O2-induced DNA breakage in mitochondria could be blocked by the treatment of MnTBAP [27]. Moreover, the peroxidase activity of Mn (Ⅲ) protoporphyrin IX-6(7)-gly-gly-his methyl ester (MnGGH) was proposed [35]. Formation of Zn-containing PPn (Zn-PPn) was reported in a Mg chelatase (bchD) mutant of Rhodobacter sphaeroides [15]. Contrary to the antioxidative effect of the above Mn-porphyrins, the seemingly opposite effect of Zn-PPn on oxidative stress was illustrated by its inhibition of heme oxygenase-1 of the tumor cell, thereby decreasing the level of bilirubin [30]. Since bilirubin is known to act as an antioxidant, Zn-PPn showed potent antitumor effect through the indirect increase of ROS in tumor cells [30]. When the heme in prostaglandin endoperoxide synthase was substituted with Mn (Ⅲ)-PPn, the substituted enzyme still showed cyclooxygenase activity but had approximately 100-fold decreased peroxidase activity compared with the native enzyme [39]. Thus, the antioxidative activities and the ROS-related functions have been demonstrated by a variety of synthetic and natural metalloporphyrin derivatives.

In this work, we examined whether any intermediate of the bacteriochlorophyll (Bch) biosynthetic pathway illustrates antioxidative activity. Interestingly, Mg-PPn, which is accumulated by the bchM (S-adenosylmethionine: Mg-PPn methyltransferase) mutant of R. sphaeroides during anaerobic growth in the presence of dimethylsulfoxide (DMSO) as a terminal electron acceptor, showed peroxidase activity. Furthermore, Mg-PPn was found freely diffusible through the membranes of E. coli and V. vulnificus. The result further corroborates that the exogenously added Mg-PPn protects the V. vulnificus katG mutant from H2O2 stress. Unlike photosensitizers such as PPn and tetracycline, Mg-PPn did not reveal any phototoxicity after exposure to UV-A light. Rather it protected E. coli in a dose-dependent manner from the killing effect by UV-A. Accordingly, the usefulness of Mg-PPn as an antioxidant and a UV block is proposed.

 

Materials and Methods

Bacterial Strains and Growth Conditions

E. coli was grown under aerobic conditions at 37℃ in Luria-Bertani (LB) medium [36]. V. vulnificus MO6-24/O was grown aerobically at 30℃ in LB medium supplemented with 2% (w/v) NaCl (LBS, pH 7.5). R. sphaeroides 2.4.1 was grown in Sistrom’s succinate-based minimal (Sis) medium [37] aerobically and anaerobically as described previously [11]. When appropriate, kanamycin (Km) and tetracycline (Tc) were added to E. coli and R. sphaeroides cultures as indicated previously [17]. Tc was added to the V. vulnificus culture atthe concentration of 2 µg/ml.

Construction of Mutant

A 580 bp DNA fragment upstream from the 83rd residue of BchM of R. sphaeroides was PCR-amplified with the forward primer (5’-GTG CGG CTC GCG GGC TGC G-3’) and reverse primer (5’-CTG CGC GCC GCG GGC CG-3’, where the SacⅡ site is in bold), and cloned into T-vector. Likewise, a 522 bp DNA fragment downstream from the 208th residue of BchM was PCR-amplified with the forward primer (5’-GTG TCC CGC GGC TTC TAC-3’, where the SacⅡ site is in bold and the mutated sequence is underlined) and reverse primer (5’-CGA GGC CCG CCG TCT GGG-3’), and cloned into T-vector. The inserted DNA fragments were isolated after digestion of the recombinant plasmids with PstⅠ (in the multicloning sites of T-vector) and SacⅡ, and then cloned into a PstⅠ site of pBluescript (Stratagene). The resulting 1.1 kb DNA fragment was cloned into a PstⅠ site of suicide plasmid pLO1 (kanamycin resistant (Kmr)) [23] to generate pLObchM. It was mobilized into R. sphaeroides as described previously [9], and the single-crossover exconjugant (Kmr) was isolated and subjected to segregation to double crossover (bchM mutant: Kms) on Sis agar plate containing sucrose (10%).

Preparation of the Biosynthetic Intermediates for Bch

Intermediates of the Bch biosynthetic pathway were purified using bchM mutant, bchFNB mutant [20], bchZ mutant [18,20], bchF mutant [18-20], bchZF mutant [18], bchC mutant [20], and bchG mutant [20]. The mutants were cultivated in Sis medium supplemented with Tween 80 (0.03%) under dark anaerobic conditions (with DMSO). Mg-PPn was extracted either from the cells of bchM mutant using acetone: methanol (7:2, by volume) [31] or from the culture supernatants using diethyl ether as performed to extract other Bch intermediates [28]. The extracted pigments, which had been dried with N2 stream [28], were dissolved in 50% ethanol prior to use. The level of Mg-PPn was determined by spectrophotometric measurements using the extinction coefficient (ε) at 419 nm (ε = 308 mM-1cm-1) [41].

Catalase Assay

Catalase activity was measured in 50 mM potassium phosphate buffer containing 20 mM H2O2 and 100 µM Mg-PPn. The activity was determined by measuring the decrease of absorbance at 240 nm, which reflects the level of H2O2 [33].

Peroxidase Assay

Peroxidase activity was performed at 30℃ in 100 mM potassium phosphate buffer (pH7.0) supplemented with 5 mM 2,4-dichlorophenol (DCP) as an electron donor, 3.2 mM 4-aminoantipyrine (AAP), and 5 mM H2O2 as described previously [26,38]. The activity was measured by monitoring the increase of optical density at 510 nm, which reflects the level of quinoneimine, a Schiff base synthesized from the reaction between AAP and the oxidized electron donor DCP. The activity, which was calculated using the extinction coefficient of quinoneimine at 510 nm (ε = 6.58 mM-1cm-1), was expressed as U (unit = µmol quinoneimine/min) per mg of metalloporphyrins such as cobalt (Co)-, copper (Cu)-, gallium (Ga)-, magnesium (Mg)-, manganese (Mn)-, nickel (Ni)-, tin (Sn)-, zinc (Zn)-, and Mg-PPn. All the metalloporphyrins were purchased from Frontier Scientific (Utah, USA).

Determination of Mg-PPn Monomethyl Ester (MPME)

E. coli DH5α and V. vulnificus MO6-24/O were transformed with a pRK415 [16] derivative, pRKbchM, which contains a 0.8 kb DNA of the bchM gene of R. sphaeroides, which extends from the 80 bp upstream of its start codon to the 37 bp downstream of the stop codon, in the same orientation as the lac promoter of the plasmid. They were grown in LB and LBS supplemented with Mg-PPn (10 µM), and cells were harvested during exponential growth (A600 at approximately 0.5). Porphyrins were extracted from the cells using acetone:methanol (7:2, by volume) [31], and further analyzed by HPLC (LC-6AD, Shimadzu, Japan) on a Synergy 4 µ Max-RP column (Phenomenex, 150 × 4.6 mm) using a fluorimetric detector (excitation wavelength of 420 nm and emission wavelength of 595 nm) as described previously [24]. The column was run using a 20 min linear gradient from 85% H2O, 15% acetonitrile, and 0.05% triethylamine (by volume) to 100% acetonitrile at a flow rate of 1 ml/min. Mg-PPn and MPME were eluted at approximately 9 and 13.5 min, respectively, under these conditions. The identity of the pigments was also confirmed by fluorescence emission spectra on a spectrofluorimeter (Quanta master QM-4/2005; PTI, USA) using an excitation wavelength of 420 nm.

Cell Survival by Mg-PPn from H2O2 Stress

V. vulnificus katG mutant [33] was grown aerobically to logarithmic phase in LBS, and then harvested during exponential growth (A600 at approximately 1.0). Cells were washed twice with phosphate-buffered saline (pH7.5) supplemented with 3% NaCl (PBS), and suspended to a final cell density of 105 colony forming unit (CFU)/ml of PBS containing 50 µM H2O2. A control experiment was done in the same buffer without H2O2. Cells were incubated at 30° with shaking, and aliquots were taken intermittently during 90 min to determine the viable counts (CFU/ml) on LBS agar plates. Relative survival (%) was expressed as the percentage of the initial count of CFU.

Phototoxicity Under Ultraviolet (UV)-Light Irradiation

The phototoxicity of Mg-PPn was determined as described previously with minor modifications [25]. E. coli DH5α was grown to logarithmic phase (A600 at approximately 0.5) in LB, and an aliquot (0.5 ml) was harvested and inoculated into LB containing tetracycline, PPn, and Mg-PPn (each at 1.2 μM or more, especially for Mg-PPn), which had been dissolved in ethanol (50%) before its addition to the medium. Ethanol only was added to the control tube at the final concentration of 0.1%. The samples were incubated at 30℃ for 10 min to allow the diffusion of treated chemicals into cells. UV-A (320-400 nm) light with the intensity of 0.045 J/cm2 or 0.54 J/cm2 was irradiated to the cells for 10 min. Cells were then diluted into LB and incubated for 20 min at 23℃ to allow the dispersion of the treated chemicals from cells prior to plating. Viable counts (CFU/ml) were determined on LB agar plates, and the relative survival (%) was expressed as the ratio of viable counts between a UV-exposed sample and an unexposed one.

 

Results and Discussion

Mg-PPn Shows Peroxidase Activity.

It has been known that the light-absorbing pigments as well as other cellular components of the photosynthetic purple-nonsulfur bacteria such as Rhodobacter capsulatus and Rhodopseudomonas palustris are beneficial to the health of fish and shrimp [3,21,32]. The solvent-extracted pigments from R. capsulatus inhibited lipid peroxidation [32]. However, irradiation of the free Bch generates singlet-state oxygen that could cause oxidative damage to cells [43] unless carotenoids dissipate the ROS energy through heat [7]. The antioxidative activity of photosynthetic bacteria has been mostly attributed to carotenoids, so the metabolic intermediates (Fig. 1) for bacteriochlorophyll biosynthesis have not been examined yet for their antioxidative activity. In this work, several Bch biosynthetic mutants of R. sphaeroides were cultured under anaerobic conditions in the presence of DMSO, and the Bch intermediates that accumulated in cells or culture supernatants were solvent-extracted and examined for antioxidative activity. The bch mutants used were as follows: bchM mutant, bchFNB mutant [20], bchZ mutant [18,20], bchF mutant [18-20], bchZF mutant [18], bchC mutant [20], and bchG mutant [20] (Fig. 1A). Among the intermediates from several bch mutants, only Mg-PPn, which is derived from the bchM mutant, showed peroxidase activity (Fig. 2). Approximately 110 U of peroxidase activity was observed with 1 mg of Mg-PPn, whereas no catalase activity was detected (data not shown). Furthermore, the peroxidase activityof Mg-PPn increased in direct proportion to its concentration (Fig. 3), yielding a specific activity of approximately 4 × 10-2 units/µM of Mg-PPn. The extracted Mg-PPn from bchM mutant showed a spectral profile that was virtually the same as that of the commercially available one (Fig. 1B).

It was examined whether the peroxidase activity was unique to the PPn chelated with Mg ion (Fig. 3). No significant peroxidase activity was found with PPn chelated with Co, Cu, Ga, Ni, and Sn in comparison with that of Mg-PPn (Fig. 3). The derivatives of PPn chelated with Zn and Mn have been known to contain some peroxidase activity [10,27,35]. However, Zn-PPn and Mn-PPn yielded 10-20% of the peroxidase activity of Mg-PPn at the most. Thus, Mg-PPn was the most effective antioxidant among the metallo-PPn examined (Fig. 3). The mechanism for the peroxidase reaction by Mg-PPn in the presence of electron donor remains to be elucidated.

Fig. 1.Biosynthetic pathway of Bch. (A) PPn, which is synthesized from δ-aminolevulinic acid (ALA), is chelated with the Fe ion by ferrochelatase (hemH) and with the Mg ion by magnesium chelatase (bchHID) to form Fe-PPn (heme) and Mg-PPn, respectively. The methylation of Mg-PPn by BchM leads to the formation of MPME, which is metabolized further to synthesize Bch. The genes illustrated code for the following enzymes: bchE, MPME oxidative cyclase; bchJ, 4-vinyl reductase; bchLNB, light-independent protochlorophyllide a reductase; bchXYZ, chlorophyllide a reductase; bchF, 3-vinyl bacteriochlorophyll hydratase; bchC, 3-desacetyl-3-hydroxyethyl bacteriochlorophyllide a dehydrogenase; bchG, bacteriochlorophyll synthase; bchP, geranylgeranyl reductase. The structure of Mg-PPn is shown at the right side of Mg-PPn of the metabolic pathway. (B) The spectral profile of the extracted Mg-PPn from bchM mutant is shown with that of the commercially available Mg-PPn.

Fig. 2.Peroxidase activity of Mg-PPn. The peroxidase activity of Mg-PPn was measured by monitoring the formation of quinoneimine at 510 nm, which is a condensation product through the formation of Schiff base between AAP and the oxidized electron donor DCP in the presence of H2O2. Control with Mg-PPn only (the first bar) is shown together with those without Mg-PPn (the second bar), H2O2 (the third bar), DCP (the fourth bar), and AAP (the fifth bar). Peroxidase activity by Mg-PPn (the sixth bar) was expressed as U (unit = µmol quinoneimine/min) per mg of Mg-PPn. The error bars correspond to the standard deviations of the means. The + stands for the inclusion of the component in the reaction mixture, whereas − stands for its omission.

Fig. 3.Peroxidase activities of metalloporphyrins. Peroxidase activity was measured using various metalloporphyrins (1 to 20 µM) including Mg-PPn. The activity was expressed as U (unit = µmol quinoneimine/min). Co, cobalt; Cu, Copper; Ga, Gallium; Mg, Magnesium; Mn, Manganese; Ni, Nickel; Sn, Tin ; Zn, Zinc.

Mg-PPn Protects Cell from H2O2.

It was determined whether Mg-PPn can freely enter the cell by diffusion through the membrane. E. coli and V. vulnificus were transformed with pRKbchM, a recombinant pRK415 carrying bchM DNA coding for the S-adenosylmethionine: Mg-PPn methyltransferase of R. sphaeroides. They were cultured aerobically in medium supplemented with Mg-PPn (10 µM). The cells were washed with PBS, and then extracted with acetone-methanol to detect Mg-PPn monomethyl ester (MPME). Both recombinant E. coli and V. vulnificus containing pRKbchM yielded MPME, which was identified by HPLC (Fig. 4). No MPME was detected with the recombinant cells containing vector plasmid only. Since BchM is a cytosolic enzyme [12], the formation of MPME by the recombinant cells containing pRKbchM clearly indicates that Mg-PPn should freely diffuse into the cell membrane.

Fig. 4.Uptake of Mg-PPn into E. coli (pRKbchM) and V. vulnificus (pRKbchM), and the fluorimetric detection of MPME. (A) E. coli (pRKbchM) and (B) V. vulnificus (pRKbchM) were cultivated in the presence of Mg-PPn (10 µM), and porphyrins were extracted from the cells with acetone: methanol (7:2, by volume). Mg-PPn and MPME were further analyzed by HPLC using a fluorimetric detector (excitation wavelength of 420 nm and emission wavelength of 595 nm). Mg-PPn was eluted at approximately 9 min and MPME at approximately 13.5 min.

Fig. 5.Effect of Mg-PPn on the survival of V. vulnificus katG mutant after H2O2 treatment. Cells, which had been cultured in LBS, were transferred to PBS (pH 7.5, 3% NaCl) containing 50 µM H2O2 in the presence (2 µM or 10 µM) or absence of Mg-PPn. Treatment of cells with Mg-PPn only (10 µM) was included as a control. Viable cell counts on LBS agar plates were done with the aliquots taken intermittently during the 90 min exposure to H2O2. The relative survival (%) was expressed as the percentage of the initial count of CFU. The error bars correspond to the standard deviations of the means.

Since Mg-PPn is found diffusible through the membrane, it was determined whether the exogenously added Mg-PPn protects cell from H2O2 stress. The katG mutant of V. vulnificus [33] was used because the bacterium has KatG, a catalase-peroxidase, only in the cytosol. Its KatE, a catalase, has a signal peptide for membrane translocation, so it is thought to be present in the periplasmic space. The katG mutant was sensitive to H2O2, where its survival decreased by more than 90% after exposure to 50 µM H2O2 for 1 h (Fig. 5). Mg-PPn protected the katG mutant in a dose-dependent way from H2O2. Thus, Mg-PPn can be used as an antioxidant through its exogenous addition to the cell. The electron donor for Mg-PPn in cell remains to be identified.

Mg-PPn Does Not Show Any Phototoxicity, But Rather Protects the Cell from UV Light.

Most porphyrin compounds are photosensitizers, demonstrating phototoxicity when exposed to UV light [13]. The phototoxic effect of Mg-PPn toward E. coli was examined after its exposure to UV-A (320~400 nm), which comprises approximately 90% of the UV light in day illumination with higher penetrability compared with UV-B (320~280 nm). No decrease in survival of E. coli by UV-A was observed in the control tube containing ethanol only. When tetracycline was irradiated with UV-A, however, more than 90% of the E. coli cells were killed. Irradiation of PPn with UV-A resulted in survival reduction by approximately 30%. On the contrary, Mg-PPn did not show any phototoxicity under these conditions (Fig. 6).

Fig. 6.No significant phototoxicity of Mg-PPn toward E. coli under UV-light stress. UV-A light (0.045 J/cm2) was irradiated to E. coli cells for 10 min in the presence of tetracycline, PPn, and Mg-PPn (1.2 µM each). A control experiment was performed with ethanol (EtOH, 0.1%) only. Viable counts were determined by plating the serial dilutions of cells on LB agar plates. Relative survival (%) was expressed as the ratio of viable counts between a UV-A-exposed sample and an unexposed sample. The error bars correspond to the standard deviations of the means.

The same phototoxicity test was repeated with 12-fold stronger UV-A (Fig. 7), where the survival of E. coli was reduced by approximately 80%. The survival of E. coli went up to approximately 40% with 1.2 µM of Mg-PPn, which had been used in Fig. 6. Moreover, approximately 90% survival was achieved by increasing Mg-PPn up to 6 µM. Thus, the phototoxicity of Mg-PPn was not found under UV-A illumination. Rather, it protects cells from UV-induced damage.

The cause of the phototoxicity of a photosensitizer is usually ascribed to oxidative stress, which is generated in the presence of O2 by its exposure to UV-A [25]. The phototoxicity of Mg-PPn was not expected, since it illustrates peroxidase activity. Moreover, bchM mutant of R sphaeroides, which accumulates Mg-PPn in cell, is not sensitive to visible light under aerobic conditions. Although the differences of peroxidase activities between Mg-PPn and other metalloporphyrins such as MnTBAP and MnMPyP remains to be determined, one remarkable property of Mg-PPn is its photoprotective function against UV-A. Accordingly, Mg-PPn can be usefully applied not only to protect cells from H2O2 stress but also from damage by UV-A. Thus, its application could be extended from an antioxidant to an ingredient in sun blocks.

Fig. 7.Photoprotective effect of Mg-PPn on E. coli from UV light. UV-A light with the intensity of 0.54 J/cm2 was irradiated to the cells for 10 min in the presence of Mg-PPn (1.2 µM or 6 µM). A control experiment was performed with ethanol (0.1%) only. Viable counts were determined by plating the serial dilutions of cells on LB agar plates. Relative survival (%) was expressed as the ratio of viable counts between a UV-A exposed sample and an unexposed one. The error bars correspond to the standard deviations of the means.

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