The FASEB Journal • Research Communication

A novel role for iron in modulating the activity andmembrane-binding ability of a trimmed soybeanlipoxygenase-1

Enrico Dainese,*,1,2 Clotilde B. Angelucci,*,1 Annalaura Sabatucci,*Vincenzo De Filippis,† Giampiero Mei,‡ and Mauro Maccarrone*,§,2

*Department of Biomedical Sciences, University of Teramo, Teramo, Italy; †Department ofPharmacological Sciences, University of Padua, Padua, Italy; ‡Department of Experimental Medicineand Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy; and §European Center forBrain Research/Santa Lucia Foundation, Rome, Italy

ABSTRACT Lipoxygenases (LOXs) are iron-contain-ing enzymes that play critical roles in plants and ani-mals. As yet, metal atom extraction, reconstitution, andsubstitution have not been successfully applied to soy-bean LOX-1 [Glycine max (L.) Merrill], a prototypemember of the LOX family that is widely used instructural and kinetic studies. Here, tryptic digestion ofnative LOX-1, used as a control, allowed us to isolatethe 60-kDa C-terminal region (termed miniLOX), thatretains the catalytically active iron in a more accessibleposition. Then, iron was removed to obtain an unprec-edented apo-miniLOX, which was reconstituted andsubstituted with different metal ions. These forms ofminiLOX were characterized vs. native LOX-1 by ki-netic analysis, near UV circular dichroism, steady-statefluorescence, and fluorescence resonance energy trans-fer. MiniLOX showed a 2-fold increase in the mem-brane-binding affinity compared with native LOX-1 anda remarkable 4-fold increase compared with apo-miniLOX (Kd�9.2�1.0, 17.9�2.0, and 45.4�4.3 �M,respectively). Furthermore, miniLOX reconstitutedwith Fe(II) or Fe(III) partially recovered its mem-brane-binding ability (Kd�21.4�2.4 and 18.9�5.5 �M,respectively), overall supporting a novel noncatalyticrole for iron in the LOX family.—Dainese, E., Ange-lucci, C. B., Sabatucci, A., De Filippis, V., Mei, M.,Maccarrone, M. A novel role for iron in modulating theactivity and membrane-binding ability of a trimmedsoybean lipoxygenase-1. FASEB J. 24, 1725–1736(2010). www.fasebj.org

Key Words: limited proteolysis � metal substitution � enzymeactivity � enzyme structure � pathogen infection

Lipoxygenases (linoleate:oxygen oxidoreductase, EC1.13.11.12; LOXs) contain one nonheme iron cofactorper enzyme molecule, which catalyzes the regio- andstereospecific dioxygenation of polyunsaturated fattyacids with one or more pentadiene systems (1, 2).Mammalian LOXs are implicated in several pathophys-iological processes (3–6), and in plants LOXs serve

critical functions such as promotion of germination,synthesis of traumatin and jasmonic acid, and responseto biotic and abiotic stress (7, 8). The substrates for theplant enzymes are generally linoleic and linolenic acids,whereas animal LOXs catalyze the conversion of ara-chidonic acid to leukotrienes and lipoxins (9) and tohydroxyl fatty acids (10).

Essential structural features of plant and animalLOXs are highly conserved, although they share only alow degree of sequence homology; thus, soybean 15-LOX-1 has been widely used as a prototype for studyingthe functional and structural properties of the LOXfamily (4). The crystal structures of soybean LOX-1 (11,12) revealed that the protein is organized into twodomains (12), a �-barrel N-terminal domain consistingof 146 aa (111 in mammals) and a larger C-terminaldomain that comprises amino acid residues 147–839,and is mainly �-helical (10). The C-terminal domaincontains the catalytically active nonheme iron (in a 1:1molar ratio with the protein) and the substrate-bindingcavity. Recently, new structural information has beenmade available through the comparison of the solutionstructure of soybean LOX-1 (13) with that of rabbit15-LOX (14), both obtained by small-angle X-ray scat-tering. Overall, the mammalian enzyme was found tohave a higher degree of motional freedom, which hasfunctional relevance for a different regulation of cata-lytic activity and membrane binding of these two LOXisoforms (15).

Interestingly, LOX activity induces irreversible dam-age to organelle membranes (16, 17), forming the basisfor the converging role of mammalian and plant LOXsin the induction of apoptosis by several unrelatedstimuli (18). In addition, LOXs are cytosolic enzymesthat have a tendency to translocate to biomembranes.

1 These authors contributed equally to this work.2 Correspondence: Department of Biomedical Sciences,

University of Teramo, Piazzo Aldo Moro n. 45, Teramo,Italy 64100. E-mail: M.M., mmaccarrone@unite.it; E.D.,edainese@unite.it

doi: 10.1096/fj.09-141390

17250892-6638/10/0024-1725 © FASEB

For instance, some mammalian LOXs, such as 5-LOX,are activated by a Ca2�-mediated translocation fromplasma membranes to the nucleus on cell stimulation(19, 20). Both plant and mammalian LOXs are alsoable to bind to artificial lipid bilayers in a Ca2�-dependent manner (20, 21). Thus, LOXs can be con-sidered a paradigm of enzymes working at the mem-brane-cytosol interface that require selective targetingand binding of the protein to the lipid surface andeffective substrate recognition (22). As a consequence,protein conformational changes possibly resulting fromremoval of prosthetic groups can be relevant in modu-lating the interfacial catalysis performed by this class ofenzymes.

Metal atom extraction, reconstitution, and substitu-tion, an approach that has been widely used to eluci-date the structure-function relationship of several met-alloenzymes and cyclooxygenases (23, 24), has not yetbeen successful with soybean LOX-1. In contrast, theextraction of iron from porcine leukocyte 12-LOX (25)and from soybean LOX-3 (26) has been reported,although the apo-forms could not be isolated, charac-terized (26), or reconstituted (25). In this context,limited proteolytic cleavage of LOX-1 into the two N-and C-terminal domains has added new important struc-tural and functional information (21). In particular, kineticanalysis of the 60-kDa C-terminal domain of LOX-1 (termedminiLOX) demonstrated that the trimmed enzyme has agreater catalytic activity than intact LOX-1 and displays ahigher binding affinity toward artificial membranes (21,27). Here, we sought to take advantage of miniLOXto investigate the effect of iron removal on theenzymatic activity and membrane-binding ability ofthe trimmed enzyme. Furthermore, we evaluated theeffects of reconstitution and metal substitution of theapo-miniLOX on its catalytic activity and membrane-binding properties, alone or in the presence of aviscogen, like glycerol, or of calcium ions. Takentogether, the reported data provide an unprece-dented characterization of the structural, kinetic,and membrane-binding properties of apo-miniLOXand its reconstituted and substituted forms, whichallowed the disclosure of a novel role for iron in thestructural stability of LOX and hence in the modu-lation of its membrane binding.

MATERIALS AND METHODS

Materials and enzymes

Linoleic (9,12-octadecadienoic) acid, N-tosyl-l-phenylalanine-chloromethyl ketone (TPCK) -treated trypsin, soybean trypsininhibitor, cocktail of protease inhibitors, protease type XIV(Pronase) from Streptomyces griseus, desferrioxamine mesylate,and FeCl3 �6H2O, FeCl2 �4H2O, CoCl2 �6H2O, NiCl2 �6H2O,and MnCl2 �H2O were purchased from Sigma-Aldrich Corp.(St. Louis, MO, USA). LOX-1 was purified from soybean[Glycine max (L.) Merrill, Williams] seeds as reported (28),and miniLOX was produced by tryptic digestion of LOX-1 asdescribed (21). In brief, 200-�l aliquots of LOX-1 (7 mg/ml

in 0.2 M Tris �HCl, pH 8.0) were treated with 20-�l aliquots ofTPCK-treated trypsin (7 mg/ml in 0.2 M Tris �HCl, pH 8.0)for 30 min at 25°C. Reactions were stopped by addition of60-�l aliquots of soybean trypsin inhibitor (7 mg/ml in 0.2 MTris �HCl, pH 8.0) (21). In a separate set of experiments,miniLOX was also obtained by Pronase digestion as follows:25-�l aliquots of LOX-1 (0.4 mg/ml in 0.2 M Tris �HCl, pH8.0) were treated with 10-�l aliquots of Pronase (0.07 mg/mlin 0.2 M Tris �HCl, pH 8.0) for 10 min at 25°C. Reactions werestopped by addition of 10-�l aliquots of cocktail of proteaseinhibitors (5 mg/ml in 0.2 M Tris �HCl, pH 8.0). MiniLOXused in this work was purified as described previously (21),with the addition of a further final step of adsorption chro-matography. For the latter step, a hydroxyapatite column(HA Ultrogel; Pall Corporation, Port Washington, NY, USA)was eluted with potassium phosphate buffer (pH 7.0) on alinear gradient (from 5 mM to 1 M) of potassium phosphate,using an automated fast protein liquid chromatography ap-paratus (AKTA Explorer System; GE Healthcare, Uppsala,Sweden). Protein concentration was determined according tothe method of Bradford (29).

Apo-miniLOX preparation

Removal of iron was performed by means of citric acid as achelator and of the competition with hydrogen ions at low pH(30, 31). All experiments were performed using iron-freewater, dialysis bags, and plastics. Aliquots (200 �l) of 100 �Mprotein solutions (in 0.2 M Tris �HCl, pH 8.0) were dialyzed inVisking tubes against 3 changes of 50 mM citrate/Na2HPO4,pH 4.4 (20 ml), for 5 h at 4°C. The dialysis buffer was gentlyagitated during incubation. At the end of dialysis, proteinsamples were analyzed for iron content by means of atomicabsorption spectroscopy. The iron content of 100 �M apo-miniLOX was below the detection limit of the instrument,indicating that �5% of miniLOX retained its metal cofactorafter dialysis. In a separate set of experiments, iron removalfrom miniLOX was accomplished also by overnight dialysisagainst 10 mM Tris �HCl, pH 7.5, with desferrioxamine as theiron-chelating agent in a 1:5 miniLOX:desferrioxamine stoi-chiometry, followed by dialysis for 48 h against the samebuffer containing 2 mM EDTA.

Metal derivatives of miniLOX

For metal substitution experiments, aliquots (200 �l) of 100�M apo-miniLOX were dialyzed against 50 mM Tris �HClbuffer, pH 7.5 (20 ml), containing 1 M NaCl and 2 mMsubstitution ion (FeCl3, FeCl2, CoCl2, NiCl2, or MnCl2).Then, reconstituted enzymes were diluted 10-fold in 1 mMsubstitution ion solution to ensure �99% metal incorpora-tion (30) and were finally dialyzed and concentrated 10-foldusing Centricon-10 ultrafiltration units (Amicon, Beverly,MA, USA). Apo-miniLOX and its reconstituted forms werethen used in enzyme activity and membrane-binding assays, aswell as in the structural studies detailed below. To solubilizeFeCl2 and FeCl3 solutions, 10% (w/v) glucose was added;under these experimental conditions no precipitates wereobserved. To remove the unspecifically bound metal, allminiLOX derivatives were dialyzed for 48 h against a 50 mMTris �HCl buffer, pH 7.5, containing 2 mM EDTA, and for afurther 24 h against the same buffer without EDTA. Inaddition, holo-miniLOX was treated with 2 mM FeCl2 orFeCl3 solutions under the same reconstitution and dialysisconditions described above to check the possible effect ofunspecifically bound iron on enzyme structure and function.

Metal analysis was performed by atomic absorption spec-troscopy with a model 5000 spectrophotometer (PerkinElmer

1726 Vol. 24 June 2010 DAINESE ET AL.The FASEB Journal � www.fasebj.org

Life and Analytical Sciences, Boston, MA, USA), equippedwith a graphite furnace or an acetylene/air burner for flameanalyses. A standard addition method was applied usingatomic absorption standard solutions of iron, copper, nickel,and cobalt from Sigma-Aldrich. Each standard was added tosolutions containing from 5 to 10 �M miniLOX. No interfer-ences were observed in metal determination under theseconditions.

Enzyme activity

LOX activity was assayed spectrophotometrically at 25°C in100 mM sodium borate buffer (pH 9.0) by recording theformation of conjugated hydroperoxides from linoleic acid at234 nm (32). Kinetic studies were performed using differentsubstrate concentrations (in the 0–120 �M range) to calcu-late the kinetic parameters. The experimental points wereanalyzed by nonlinear regression through the Sigma Plot2000 program (SPSS Science, Chicago, IL, USA). Lag phasevalues of the dioxygenation of 90 �M linoleic acid by recon-stituted miniLOXs (7.5 nM) were determined as reportedpreviously (33).

Near-UV circular dichroism (CD) and steady-statefluorescence measurements

Near-UV CD spectra were recorded on a Jasco-710 spectropo-larimeter (Jasco, Easton, MD, USA), at 20°C, using a 0.5-cmquartz cuvette. Steady-state fluorescence spectra were re-corded at 25°C using an LSB50 fluorometer (PerkinElmerLife and Analytical Sciences). At the excitation wavelength of284 nm, emission spectra were recorded between 300 and 400nm, using a 10 � 2 mm pathlength quartz fluorescencemicrocuvette (Hellma GmbH & Co., Mullheim/Baden, Ger-many).

Liposome preparation

Large unilamellar vesicles (LUVs) were prepared with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Lyophilizedlipids were dissolved in a small volume of ethanol, which wassubsequently evaporated under nitrogen at T � Tm (T�55°C;main lipid transition temperature Tm�41°C) to form a thin-layer lipid film around the walls of a glass tube. The dry lipidfilms were then resuspended in the proper buffer at T � Tm.The final concentration of lipids in the buffer was 1 mM. Toobtain uniformly sized LUVs, lipid extrusion was performed byfiltering the samples 21 times through 100-nm-pore polycarbon-ate membranes in a LiposoFast extruder (Avestin, Ottawa, ON,Canada), kept at a temperature �Tm. Dansyl-liposomes used influorescence resonance energy transfer (FRET) studies con-tained 5% (w/w) N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (dansyl-DHPE; triethylammonium salt) from Molecular Probes(Eugene, OR, USA).

FRET studies of Ca2�-dependent membrane binding

Membrane insertion of native LOX-1, miniLOX, apo-miniLOX, and Fe(II)- and Fe(III)-reconstituted forms wasmonitored by the loss in Trp fluorescence and the corre-sponding increase in dansyl-DHPE fluorescence on the addi-tion of Ca2�. Solutions contained dansyl-liposomes at aconcentration of 260 �M and each enzyme at a proteinconcentration of 1.2 �M. Membrane affinity was determinedby monitoring the decrease in Trp fluorescence on theaddition of calcium ions to solutions of LUVs, in which

dansyl-DHPE was incorporated at 5% (w/w) concentration.Each enzyme was used at 0.4 �M, and the liposome concen-tration varied between 10 and 80 �M in a sample volume of100 �l. Solutions containing freshly prepared protein dansyl-DHPE liposomes were analyzed for each data point. Emissionspectra (exc�295 nm and em�320–420 nm) were recordedin the presence of 2 mM EDTA and rescanned after theaddition of 4 mM CaCl2. Data were plotted as fractional lossof Trp fluorescence (�F/�Fmax) vs. liposome concentration,according to a published procedure (34). Experimental datawere analyzed by nonlinear regression through a hyperbolicbinding isotherm, using the Sigma Plot 2000 program (SPSSScience).

Statistical analysis

Data reported in this article are the means � sd of �4independent determinations, each performed in triplicate.Statistical analysis was performed by the nonparametricMann-Whitney U test, with analysis of experimental data bymeans of the InStat 3 program (GraphPad Software forScience, San Diego, CA, USA).

RESULTS

Functional properties of native LOX-1 and miniLOX

To obtain larger amounts of the trimmed enzymesuitable for metal removal and substitution experi-ments, we improved the final step of the availablemethod for miniLOX purification (21). The 60-kDafragment corresponding to miniLOX was purified,taking into account the fact that the trimmed enzymehas larger hydrophobic regions exposed to the solventthan the native enzyme. On this basis, chromatographicseparation on a hydroxyapatite matrix was added as afurther step. This improved purification allowed us toisolate a fraction eluting at 350 mM potassium phos-phate (Fig. 1) that represented a highly homogeneous

Figure 1. Final step of miniLOX purification. Eluent B: potas-sium phosphate with a linear gradient from 5 mM to 1 M. Mainpeak, eluting at 35% B, corresponded to pure miniLOX asassessed by 12% SDS-PAGE (inset). Inset: lane 1, miniLOX; lane2, low-molecular-weight markers.

1727CHARACTERIZATION OF APO- AND METAL-SUBSTITUTED FORMS OF MINILOX

miniLOX preparation (Fig. 1, inset), purified at a 100�M concentration. This miniLOX was used in furtherbiochemical assays.

The dependence of miniLOX activity on substrateconcentration was shown to follow typical Michaelis-Menten kinetics, yielding apparent Km and kcat values of24.0 � 2.7 �M and 807.0 � 15.5 s 1 (Table 1). Thecatalytic efficiency (expressed as the specificity con-stant, s�kcat/Km) of miniLOX is 3-fold higher thanthat of full-length LOX-1, in accordance with ourprevious results (21). Interestingly, the Km value ofminiLOX increased by 2-fold compared with that ofLOX-1, whereas the kcat value increased by 6-fold(Table 1). Therefore, it seems that removal of theN-terminal domain slightly reduces the affinity of theenzyme for the substrate, but it enhances to a remark-able extent its catalytic activity. These findings are fullyconsistent with the hypothesis that removal of theN-terminal domain facilitates substrate penetrationinto the catalytic pocket as well as product release fromthe active site of the trimmed enzyme. To furthersupport this hypothesis, we compared the kinetic prop-erties of LOX-1 and miniLOX in the presence of 10%glycerol. We found that this viscogen had a very differ-ent effect on the native and the trimmed enzymes. Infact, glycerol significantly inhibited LOX-1 activity(Fig. 2A), yielding apparent Km and kcat values of 11.4 �1.2 �M and 58.0 � 3.5 s 1, respectively, and hence ans value of 5.09 � 0.70 � 106 M 1 �s 1; the latterparameter was much lower (50%) than the value(11.20�0.18�106 M 1 �s 1) calculated in the absenceof the viscogen (Table 1). Instead, glycerol did notaffect the activity of miniLOX, which showed the sameMichaelis-Menten kinetics in the presence or in theabsence of this compound (Fig. 2B). Incidentally, thekinetic data obtained for native LOX-1 are in agree-ment with previous studies, suggesting that viscogenscould play a role in hindering the access of the lipidsubstrate to the active site and in making productrelease more difficult (35).

Preparation and characterization of metal-substitutedminiLOX forms

The preparation of highly monodisperse miniLOXsolutions allowed us to set up specific protocols suitablefor iron removal from the active site and for reconsti-

tution and substitution of the enzyme with differentmetals. Subsequently, reconstituted miniLOX formswere characterized by atomic absorption spectroscopyto evaluate the metal/protein molar ratios (Table 2). Inaddition, specific activity of the different miniLOXforms was calculated to ascertain the amount of recon-stituted proteins with a functional active site (Fig. 3). Inpreliminary experiments it was found that dialysis at pH4.4 was unable to reduce enzyme activity of nativeLOX-1 (data not shown), whereas it minimized that ofminiLOX, down to 2.5% of the untreated enzyme(Fig. 3). Loss of activity was due to iron removal, andindeed iron content determination showed that 100�M miniLOX retained �0.5 �M iron after dialysis(Table 2). Therefore, the use of an iron chelator suchas citric acid and the competition with hydrogen ions atlow pH (30, 31) were suitable conditions to generate avirtually inactive apo-form of miniLOX. Conversely, the

Figure 2. Kinetic properties of native LOX-1 (A) andminiLOX (B) in the presence (f– – –f) or in the absence(E——E) of 10% glycerol.

TABLE 1. Kinetic parameters of miniLOX and its reconstituted/substituted forms, compared with those of native soybean LOX-1

Enzyme Km (�M) Vmax (�M �min 1) kcat (s 1) kcat/Km (M 1 � s 1)

Native LOX-1 10.9 � 3.1 55.0 � 5.0 122.0 � 1.8 11.20 � 0.18 � 106

MiniLOX 24.0 � 2.7 363.0 � 8.0 807.0 � 15.5 33.60 � 0.13 � 106

Apo-miniLOX ND ND ND NDMiniLOX-Fe(II) 27.0 � 0.9 79.3 � 0.8 176.2 � 1.7 6.50 � 0.04 � 106

MiniLOX-Fe(III) 30.5 � 0.6 35.6 � 13.0 79.3 � 28.5 2.60 � 0.38 � 106

MiniLOX-Co(II) 33.3 � 0.8 24.2 � 5.7 54.0 � 10.8 1.60 � 0.22 � 106

MiniLOX-Ni(II) 36.2 � 1.2 6.9 � 2.0 15.3 � 4.3 0.40 � 0.13 � 106

Data are means � sd. ND, not detectable.

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native enzyme was found to be refractory to ironextraction, in line with previous reports (25, 26). Inter-estingly, starting from native LOX-1 preparations andusing Pronase (i.e., a mix of proteases containing atrypsin-like activity from S. griseus), we were able toobtain a trimmed LOX-1 with the same molecular massand catalytic activity as miniLOX (Fig. 4). Pronasedigestion of LOX-1 also generated a second band (30kDa), much like that produced by trypsin digestion(21). In addition, natural chelators such as the sid-erophore desferrioxamine released from S. griseus andother gram-positive bacteria (36) allowed partial re-moval of iron and a reduction of catalytic activity downto 30% of controls (Fig. 4).

We took advantage of the efficient metal removalobtained with citric acid and used apo-miniLOX toperform metal reconstitution experiments. A partialrecovery (30%) of enzymatic activity was obtained ontreatment with Fe(III), and a significantly higher recov-ery (60%) was obtained on treatment with Fe(II)(Fig. 3). Similar results were reported for soybeanLOX-3, whose catalytic activity was reconstituted fromthe apoenzyme (26). In addition, reconstitution of

apo-miniLOX with Co(II) led to 20% recovery ofLOX activity, whereas reconstitution with Ni(II) yieldedonly a negligible (6%) recovery (Fig. 3). Reconstitu-tion of apo-miniLOX with Mn(II) under the sameexperimental conditions yielded almost undetectableactivity (Fig. 3), possibly due to the low sequenceidentity between the metal center of fungal Mn(II)-LOX and that of soybean Fe(II)-LOX (37).

Specific activity of the reconstituted/substitutedforms of miniLOX was compared to the pseudo-LOXactivity of the corresponding free metals in solution,tested at the same concentrations used in the reconsti-tution/substitution experiments. No catalytic activitycould be detected in the presence of any of the freemetals in solution (Fig. 3).

The catalytic activity of apo-miniLOX forms reconsti-tuted with Fe(II) or Fe(III) or substituted with Co(II)and Ni(II) showed a substrate dependence typical ofMichaelis-Menten kinetics, yielding the apparent Kmand kcat values reported in Table 1. Our results sug-gested that reconstitution with Fe(II) led to an enzymesolution with a catalytic efficiency (s) 5-fold lowerthan that of miniLOX. On the other hand, miniLOXsolutions reconstituted with Fe(III) or substituted withCo(II) or Ni(II) showed a further drop in the catalyticefficiency, 10- to 100-fold lower than that of holo-miniLOX preparations (Table 1). This dramatic reduc-tion in enzyme activity could be due to the differentstereoelectronic properties of the substituting metalions, which can affect their binding to the active site,leading overall to a different reconstituting efficiency.Indeed, although the protein-metal stoichiometry indi-cated the presence of one metal ion bound per protein

TABLE 2. Metal/protein ratios of holo-miniLOX,apo-miniLOX, and metal-reconstituted/substituted forms ofminiLOX, calculated by atomic absorption spectrometry

Sample Metal/protein (mol/mol)

MiniLOX 1.0Apo-miniLOX �0.005MiniLOX-Fe(II) 1.2MiniLOX-Fe(III) 1.3MiniLOX-Co(II) 1.0MiniLOX-Ni(II) 1.1

Figure 3. Relative enzymatic activities of apo-miniLOX and ofthe different metal-reconstituted and substituted forms withrespect to miniLOX (100%�5.6�0.3 �M/min). Nonenzy-matic activities, possibly due to the metal ions alone, are alsoshown.

Figure 4. Enzymatic activities of trimmed LOX-1 (miniLOX)generated by Pronase from S. griseus, and of the partialapo-form obtained by treatment with the natural iron chela-tor desferrioxamine (100%�5.8�0.3 �M/min). Inset: lane 1,low-molecular-weight markers; lane 2, fragments obtained byPronase digestion of LOX-1. Arrow in lane 2 indicatesminiLOX (see text for details).

1729CHARACTERIZATION OF APO- AND METAL-SUBSTITUTED FORMS OF MINILOX

molecule, in the different preparations metal atomsmight not be properly coordinated in the active site. Inthis context, the similar Km values of ferrous and ferricminiLOXs and the lower kcat value of the latter enzymecompared with that of the former (Table 1) seem tosuggest that Fe(II) allowed reconstitution of the ho-loenzyme with a better yield than Fe(III). In addition,we found that the lag phase of linoleate dioxygenationby Fe(II)- and Fe(III)-miniLOXs was 5 s in both casesand that addition of Fe(II) to the reconstitutedminiLOX-Fe(III) allowed the same activity as miniLOX-Fe(II) to be reached (data not shown). It should berecalled that an higher extent of reconstitution whenFe(II) was used instead of Fe(III) has been alreadyreported for other protein systems, such as tyrosinehydroxylase (38–40).

Structural properties of miniLOX and itsreconstituted and substituted forms

The structural properties of miniLOX, apo-miniLOX,and its reconstituted and substituted forms were investi-gated by fluorescence spectroscopy (Fig. 5) and by CD inthe near-UV region (Fig. 6). In keeping with previousdata (21), the emission maximum (max) ofminiLOX was slightly red-shifted (from 337 to 339nm), and, more important, its fluorescence intensitywas remarkably decreased (by 40%) compared withthat of native LOX-1 (Fig. 5). These differencescannot be assigned solely to the different content of Tyrand Trp residues in the full-length vs. the trimmedenzyme (i.e., 37 Tyr and 14 Trp vs. 33 Tyr and 12 Trp)(27). Instead, it seems more likely that the fluorescencechanges might reflect higher conformational flexibilityand enhanced polarity of the chemical environmentsurrounding Trp residues in miniLOX (41). Further-more, compared with miniLOX the apo-form displays a

greater shift of the emission to longer max (i.e., 343nm) and a further decrease (10%) in fluorescenceintensity. These observations indicate that removal ofthe metal ion induces a more relaxed structure inapo-miniLOX (Fig. 5). Interestingly, emission spectraof Fe(II)- but not of Fe(III)-reconstituted forms,showed a small blue shift with respect to the apo-enzyme, reaching a value more similar to that ofminiLOX (Fig. 5). This finding is suggestive of asignificant, albeit partial, extent of recovery of thenative-like protein conformation. On the other hand,Ni(II)- and Co(II)-substituted forms of miniLOXshowed a value of max of 343 nm, identical to that ofthe apo-form (Fig. 5).

To further ascertain whether miniLOX undergoesconformational changes on metal removal and substi-tution, CD spectra of native LOX-1 and holo- andapo-miniLOX derivatives were recorded in the near-UVregion (Fig. 6). For comparison, the CD spectrum ofnative LOX-1 is also reported. This spectrum wascharacterized by extensive fine structure in the 255–305nm region, with two prominent and positive bands at288 and 292 nm, assigned to the contribution of Trpresidues, and a distinct band at 280 nm, contributed byTyr residues. The absorption of phenylalanines ap-peared as two shallow bands between 255 and 268 nm.Removal of the N-terminal domain (i.e., residues1–277), caused remarkable changes in both shape andsignal intensity of the CD spectrum of holo-miniLOX.In particular, the contribution of Trp residues nowappeared as a shoulder at 290 nm, with a negative bandat 298 nm. The latter band probably arose from thealgebraic sum of positive (i.e., 1Lb transition) andnegative (i.e., 1La transition) contributions of Trp resi-dues embedded in different chemical environments(42, 43). On removal of the metal ion, a distinct banddue to Trp absorption at 290 nm was still present,whereas the contribution of Tyr residues was dramati-

Figure 5. Comparison of fluorescence emission spectra ofnative LOX-1 (dotted black), miniLOX (straight black), apo-miniLOX (red), miniLOX-Fe(II) (cyan), miniLOX-Fe(III)(blue), miniLOX-Ni(II) (pink), and miniLOX-Co(II) (green).a.u., arbitrary units.

Figure 6. Comparison of the near-UV CD spectra of LOX-1(dotted black), miniLOX (straight black), apo-miniLOX (red),miniLOX-Fe(II) (cyan), miniLOX-Fe(III) (blue), miniLOX-Ni(II) (pink), and miniLOX-Co(II) (green).

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cally reduced, indicating that the apo-form is in a morerelaxed conformation.

The CD spectra of the Fe(II)- and Fe(III)-reconsti-tuted miniLOX proteins showed some recovery of thespectral features distinctive for holo-miniLOX, whereasthe spectra of the Ni(II)- and Co(II)-reconstitutedforms were remarkably different, with a negative signalthroughout the wavelength range explored, indicatingthat the overall protein conformation was altered onmetal binding. However, possible spectroscopic effects,due to the intrinsic absorption of Ni(II) and Co(II) inthe protein complexes, cannot be ruled out (44, 45).These findings compare favorably with functional activ-ity data.

Binding of miniLOX and its reconstituted forms tomembranes

To evaluate whether the observed conformationalchanges due to metal ion bound to the active sitecould alter the membrane-binding properties of theenzyme, we analyzed the interaction of the differentminiLOX forms with synthetic vesicles. To this aim,we measured the Ca2�-regulated membrane-bindingability of LOX-1, miniLOX, apo-miniLOX, and thedifferent reconstituted forms as the increase in thefluorescence emission of a dansyl-DHPE probe, pre-viously incorporated into DPPC liposomes (dansyl-liposomes). This approach exploits the well-knownresonance energy transfer from the Trp residues ofthe protein (i.e., the energy donors) and the dansylmoiety of the derivatized liposomes (i.e., the energyacceptors) (46, 47). Ca2�-mediated membrane inser-tion was first evaluated by monitoring the increase influorescence intensity at 517 nm on the addition ofcalcium ions to the different enzyme-dansyl liposomemixtures (34). Interestingly, miniLOX (Fig. 7B) ex-hibited a higher increase (20%) in fluorescenceintensity compared with native LOX-1 (Fig. 7A). Thisobservation indicates higher membrane-binding abil-ity of the trimmed enzyme, thus extending previousdata based on ELISAs (21). In addition, apo-miniLOX (Fig. 7C) showed an 60% reduction inthe fluorescence signal, suggesting that metal re-

moval strongly impairs the membrane-binding abilityof the enzyme.

Next, we evaluated the parameters characterizingthe Ca2�-dependent membrane binding of soybeanLOX-1, miniLOX, and the different forms of thetrimmed enzyme by measuring Trp quenching atdifferent concentrations of dansyl-liposomes. In-deed, reporting the fraction of Trp fluorescencequenched in the presence of Ca2� as a function oflipid concentration (see Materials and Methods)allowed building up of binding isotherms (Fig. 8). Inaddition, the dissociation constant values (Kd) of thedifferent forms of the enzyme (Table 3) could becalculated by nonlinear regression analysis. All of thedifferent LOX forms yielded similar values of �Fmax,suggesting similar saturating behavior. However, thehyperbolic binding curves, resulting from the plot ofthe fraction of Trp fluorescence quenched in thepresence of Ca2� vs. the dansyl-liposome concentra-tion clearly showed that miniLOX raises the satura-

Figure 7. Differences in the fluorescence emission intensity of the dansyl-DHPE probe incorporated into DPPC liposomes, inthe absence (——) or in the presence (– – –) of Ca2�. A) LOX-1. B) MiniLOX. C) Apo-miniLOX. a.u., arbitrary units.

Figure 8. Ca2�-dependent membrane binding affinity ofsoybean LOX-1 analyzed as Trp FRET quenching at differentconcentrations of dansyl-liposomes. These plots allowed us tocalculate by nonlinear regression analysis the values of thedissociation constant, Kd (Table 3), of the different forms [F,LOX-1; �, miniLOX; �, apo-miniLOX; E, miniLOX-Fe(II);Œ, miniLOX-Fe(III)]. a.u., arbitrary units.

1731CHARACTERIZATION OF APO- AND METAL-SUBSTITUTED FORMS OF MINILOX

tion threshold at a lower liposome concentrationwith respect to the native form (Fig. 8). On the otherhand, apo-miniLOX showed a binding isotherm typ-ical of a protein with low affinity for membranes.Interestingly, both Fe(II)- and Fe(III)-reconstitutedforms showed partial recovery of the membrane-binding affinity, which appeared closer to that ofnative LOX-1 (Fig. 8). Consequently, the parametersobtained by fitting the curve, summarized in Table 3,indicated that the trimmed enzyme has a reductionin Kd value of 50% (from 17.9 to 9.2 �M) withrespect to the native LOX-1 (Table 3), which meansa 2-fold increase in the membrane-binding affinityof miniLOX. In addition, apo-miniLOX showed anincrease in Kd value (up to 45.4�4.3 �M), suggestiveof a large loss of membrane-binding affinity on ironremoval. Furthermore, Fe(II)- and Fe(III)-reconstitutedforms of miniLOX yielded values of Kd similar to that ofthe native LOX-1 but higher than that of miniLOX,indicating a partial recovery of the membrane-bindingability of these two forms of miniLOX.

On a final note, the addition of ferrous or ferric iron toholo-miniLOX under the same conditions used for recon-stitution and dialysis of the apo-enzyme was found not tohave an effect on catalytic activity, spectral properties, andmembrane-binding ability of the protein (not shown). Aparsimonious interpretation of these data is that unspe-cifically bound iron (if any) does not have any majoreffect on the structural and functional properties ofminiLOX.

DISCUSSION

The crystal structure of LOX-1 shows that iron isburied within the enzyme fold and it is connectedwith the surface only by a narrow channel lined withhydrophobic amino acid residues (12, 48). There-fore, enhanced catalytic activity of miniLOX can bereasonably explained on the basis of the high-resolution structural data of the full-length LOX-1(Protein Data Bank codes: 2sbl and 1f8n) (11, 12). Inparticular, the enzyme structure at 2.6 Å (11) de-scribed only one putative access for the substrate (theso-called cavity II), that is lined by 46 residues and islocated at the opposite end to the N-terminal do-main. However, subsequent work (12), which deter-

mined the structure of LOX-1 to a resolution of 1.4Å, described cavity II, which was subdivided intocavity IIa and IIb, with more detail. From bothstructural studies it appeared evident that cavity II isinside the C-terminal domain but also comprisesresidues lining the region of the cavity proximal tothe active site Fe. Interestingly, some of these resi-dues (i.e., Leu255, Glu256, Gly258, and Thr259) areremoved by trypsinolytic digestion at Lys277 thatgenerates miniLOX (Fig. 9). Furthermore, an alter-native route for substrate access was suggested at theopposite end of cavity II (12), but the structural datacould not support conclusive statements about theactive site, because it was possibly closed off in thecrystals (49). Figure 9 also shows the residues (Ile553,Leu546, and Leu754) of cavity II close to iron (11, 12)and involved in substrate binding (50). Taken to-gether, these data clearly indicate that on trypsindigestion, which removes not only the N-terminaldomain but also two �-helices belonging to theC-terminal region, one of which occludes access toiron (Fig. 9), the active site of LOX-1 is moreaccessible. Furthermore, the higher catalytic effi-ciency of miniLOX compared with that of LOX-1 isin line also with the available structural informationderived from crystallographic and spectroscopic data(1, 12, 51, 52). Indeed, the difference between thecoordination number of Fe(II) and Fe(III) sites inthe presence of the substrate has been referred to as“coordination flexibility” of LOX-1 and is thought toinvolve Asn694 (53), thus affecting kcat values. There-fore, the increased kcat value of miniLOX with re-spect to LOX-1 can be attributed also to a morerelaxed structure of the trimmed enzyme, leading toa more flexible coordination of Asn694 to the iron inthe active site (Fig. 9).

The structure of soybean LOX-1 in solution clearlydemonstrates that the protein is very compact andstable (13), ruling out any swinging movement of theN-terminal �-barrel domain with respect to the C-terminal domain. Furthermore, very recently a struc-tural comparison of the plant and mammalian LOXshas demonstrated that temperature-induced struc-tural alterations of the two enzymes are different. Inparticular, soybean LOX-1 shows a higher overallstability and a lower tendency to form aggregates insolution compared with rabbit 15-LOX (15). In thiscontext, we previously demonstrated that glycerol isable to prevent aggregation of LOX-1 in solution(13). In fact, it is well known that glycerol stabilizesproteins with large hydrophobic patches exposed tothe solvent (54), yielding a viscosity that induces apreferential hydration of proteins (55).

Therefore, the inhibition of LOX-1, but not ofminiLOX, activity by glycerol seems to indicate thatthe trimmed enzyme does not preserve the native-likestructure of the cavity IIa connecting the active site tothe protein surface. Another reasonable interpreta-tion for the present results is that in the presence ofglycerol the binding of linoleic acid is a less rate-

TABLE 3. Comparison of membrane-binding affinity of nativesoybean LOX-1 and of holo-miniLOX, apo-miniLOX, andFe(II)- or Fe(III)-reconstituted forms of miniLOX

Protein form Kd (�M)

LOX-1 17.9 � 2.0MiniLOX 9.2 � 1.0Apo-miniLOX 45.4 � 4.3MiniLOX-Fe(II) 21.4 � 2.4MiniLOX-Fe(III) 18.9 � 5.5

Data are means � sd.

1732 Vol. 24 June 2010 DAINESE ET AL.The FASEB Journal � www.fasebj.org

limiting step for miniLOX than for LOX-1, becauseof the different structures of these enzymes.

So far, owing to the presence of an highly buriedactive site, iron extraction and substitution withother metal cofactors has not been successful withnative LOX-1. Therefore, it could be surmised thatiron release from the active site of native LOX-1would require a considerable rearrangement of pro-tein conformation. Here, we took advantage ofminiLOX, the trimmed form of soybean LOX-1, thatshows larger accessibility of the active site to thesolvent, potentially representing a more suitable pro-tein for iron extraction and for reconstitution/substitution with vicariant metals. In fact, we suc-ceeded in isolating an apo-form of soybean miniLOXthat allowed us to identify a novel role of metal ion inenzyme binding to membranes. In particular, ourfunctional and structural analyses clearly show thatthe removal of iron yields an inactive form ofminiLOX, with a looser and more relaxed conforma-tion. Although the presence of disulfide bonds pre-serves the native arrangement of domains in theapo-form of various enzymes (56 –58), in the case of

miniLOX the absence of an interface between theN-terminal and C-terminal regions and of any cova-lent bond seems to make the contribution of iron toprotein stabilization indispensable. Therefore, ourresults suggest that beyond catalytic activity, the ironion plays a crucial role in the structural stability ofthe enzyme.

Interestingly, among the reconstituted forms ofminiLOX a better recovery of enzymatic activity wasobserved on Fe(II) than on Fe(III) addition (Fig. 3).However, once enzyme molecules are reconstitutedwith ferrous or ferric iron and catalytic activity (in-volving multiple turnovers) is measured, thereshould be no difference between the two. Here, itshould be noted that the similarity of Km values forferrous and ferric miniLOXs, the ability of Fe(II) toenhance the activity of miniLOX-Fe(III) up to that ofthe Fe(II)-reconstituted enzyme, and the similar lagphases of the two enzymes seem to support a fullyfunctional active site in both forms. In particular, thelatter observation suggests that iron can freely cyclebetween the Fe(II) and Fe(III) oxidation states in thereconstituted enzymes, as it does in miniLOX and

Figure 9. Details of the LOX-1 active site (Protein Data Bank file: 2sbl) where iron (magenta) is surrounded by Asn694, Ile553,Leu546, Leu754, and the C-terminal carboxylate Ile839. Tryptic cleavage at Lys277 removes an �-helix (yellow), containing someresidues lining the cavity IIa (i.e., Leu255, Glu256, Gly258, and Thr259), possibly increasing the mobility of Asn694.

1733CHARACTERIZATION OF APO- AND METAL-SUBSTITUTED FORMS OF MINILOX

LOX-1. Therefore, the different catalytic activity offerrous and ferric miniLOXs seems to be due to adifferent reconstitution yield when iron is used at twodifferent oxidation states (38 – 40). In general, it isnot unprecedented that holoenzymes are reconsti-tuted with a yield well below 100% starting from thecorresponding apo-forms (59). Furthermore, thedramatic reduction of activity of miniLOX-Ni(II) andof miniLOX-Co(II) can be caused by different yieldsof the reconstitution process, owing to the distinctmetal properties.

The structural role of metal ions within the activesite can result in overall conformational changes ofthe apo-form that could impair not only the catalyticactivity, but also the ability to work at the membrane-cytosol interface. In this context, a functional role ofCa2�-dependent binding to intracellular membranesduring cell stimulation has been demonstrated formammalian 12- and 15-LOXs (10, 20). In addition,plant cells can be induced by different elicitors andagents to increase the LOX-catalyzed conversion oflinoleic acid to jasmonic acid, through an increase inthe intracellular Ca2� concentration (60). This func-tion is probably modulated by membrane binding,which is Ca2�-dependent (15, 61). The association ofLOXs with membranes is a complex process that seems tobe driven mainly by hydrophobic interactions betweensurface-exposed nonpolar amino acids and membranelipids. In contrast, calcium ions support membrane bind-ing, presumably by forming salt bridges between acidicsurface amino acids and negatively charged constituentsof membrane phospholipids (5). In this context, struc-tural modeling of soybean LOX-1 suggested the exis-tence of two independent Ca2� binding sites, one inthe N-terminal �-barrel domain (Glu21, Glu106, andGlu179) and the other one in the C-terminal catalyticdomain (Glu673, Asp674, and Glu677) (61). The 2-foldincrease in the membrane-binding ability of thetrimmed enzyme seems to fit with this picture. In-deed, despite the absence of the N-terminal region,which contains at least one binding site for Ca2�, thelarger hydrophobic regions of miniLOX comparedwith those of LOX-1 seem to sustain a significantincrease in membrane affinity. More interestingly,metal removal not only alters the catalytic activity ofthe enzyme but also impairs its membrane-bindingability. This role for iron in LOX is rather unprece-dented. Overall, our findings support the hypothesisthat iron removal induces a global conformationalchange starting from the iron-coordinating residues(Fig. 9) and then leads to a more relaxed structure,possibly by altering the correct conformation ofCa2�-binding sites. Thus, the presence of iron in theactive site with a proper coordination geometryseems to stabilize a protein conformation that ismore competent for the selective targeting and bind-ing to the membrane surface, thus allowing moreeffective substrate recognition by the enzyme.

Furthermore, it seems noteworthy that theminiLOX generated in vitro by tryptic digestion

might be relevant in vivo also. In fact, trypsin-likeproteases have been identified in plant pathogens(62), and a trimmed LOX has been found in acellular context (63). Moreover, gram-positive soilbacteria such as Streptomyces protect soybeans againstinfection (64, 65). Here we show that proteases fromthe latter bacteria can cleave soybean LOX-1 to forma trimmed enzyme that resembles miniLOX. In ad-dition, natural siderophores released from Streptomy-ces can remove iron from the trimmed LOX-1, thussuggesting that limited proteolysis and iron removalmight be part of an arsenal that bacteria can use tointeract with plants. Incidentally, Streptomyces can alsoproduce authentic LOX inhibitors (66).

Finally, it seems noteworthy that the proteolyticremoval of an N-terminal fragment to open up thesubstrate entrance has already been reported in vitroand proposed in vivo in hemocyanin (67, 68). There-fore, limited proteolysis might represent a moregeneral paradigm for post-translational modifica-tions that generate enzyme forms with distinct bio-logical activities.

The authors thank Professor Alessandro Finazzi-Agro(University of Rome Tor Vergata, Rome, Italy) for hiscontinuing support. The kind assistance of Dr. Maria LuisaSalucci (University of L’Aquila, L’Aquila, Italy) in thepreliminary experiments on apo-miniLOX isolation andthat of Dr. Annamaria Draghi, Dr. Daniela Galla, and Dr.Paolo Di Muro (University of Padua, Padua, Italy) in CDand atomic absorption measurements, respectively, aregratefully acknowledged. This work was supported by Min-istero dell’Universita e della Ricerca (PRIN 2005) and byMinistero della Salute (Progetto Ricerca Corrente, IZS LT08/07 RC to E.D.), by the Italian Space Agency (ASI)(MoMa contract 2006), and by Fondazione TERCAS(project 2005–2008 to M.M.).

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Received for publication August 31, 2009.Accepted for publication December 17, 2009.

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