Protein oxidation in plant mitochondria detected as oxidized tryptophan

Free Radical Biology & Medicine 40 (2006) 430 – 435 www.elsevier.com/locate/freeradbiomed

Original Contribution

Protein oxidation in plant mitochondria detected as oxidized tryptophan Ian M. Møller a,*, Brian K. Kristensen b a

Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Protein Characterization, Novo Nordisk A/S, Hagedornsvej 1, DK-2820 Gentofte, Denmark Received 1 August 2005; revised 20 August 2005; accepted 22 August 2005 Available online 9 November 2005

Abstract The formation of N-formylkynurenine by dioxygenation of tryptophan was detected in peptides from rice leaf and potato tuber mitochondria. Proteins in matrix and membrane fractions were separated by two-dimensional gel electrophoresis and identified using a Q-TOF mass spectrometer. N-Formylkynurenine was detected in 29 peptides representing 17 different proteins. With one exception, the oxidation-sensitive aconitase, all of these proteins were either redox active themselves or subunits in redox-active enzyme complexes. The same site was modified in (i) several adjacent spots containing the P protein of the glycine decarboxylase complex, (ii) two different isoforms of the mitochondrial processing peptidase in complex III, and (iii) the same tryptophan residues in Mn – superoxide dismutase in both rice and potato mitochondria. This indicates that Trp oxidation is a selective process. D 2005 Elsevier Inc. All rights reserved. Keywords: Mitochondria; Mass spectrometry; Oxidative stress; Protein oxidation; Tryptophan; N-Formylkynurenine; Glycine decarboxylase; Complex III; Mitochondrial processing peptidase; Superoxide dismutase

Introduction The formation of reactive oxygen species (ROS) and, as a result, a range of other reactive compounds, is an unavoidable consequence of aerobic metabolism. These reactive compounds can react with and modify proteins, lipids, and DNA [1 –3]. Such modifications are probably involved in causing degenerative diseases and aging in humans [4]. The mitochondrion is the major site of ROS formation in mammalian cells [5] and in nonphotosynthesising plant cells [6– 8]. As a consequence, the mitochondria appear to contain more oxidatively modified (carbonylated) proteins than the chloroplasts and peroxisomes in the same tissue [9]. Despite this there is very little information about oxidative modifications of specific mitochondrial proteins and virtually none about the possible consequences of such modifications [5,10]. Abbreviations: BN, Blue Native; IEF, isoelectric focusing; GDC, glycine decarboxylase complex; LC-MS/MS, liquid chromatography/tandem mass spectrometry; MPP, mitochondrial processing peptidase; MS, mass spectrometry; MS/MS, tandem MS; PAGE, polyacrylamide gel electrophoresis; PTM, posttranslational modification; ROS, reactive oxygen species; SOD, superoxide dismutase. * Corresponding author. Fax: +45 3528 3460. E-mail address: [email protected] (I.M. Møller). 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.08.036

Taylor et al. [11] identified 51 peptides in human heart mitochondria containing N-formylkynurenine, a breakdown product of tryptophan caused by dioxygenation and ring breakage. The 51 peptides belonged to 39 different proteins, many of which are subunits of the respiratory complexes while others are redox enzymes, carriers, etc. Kristensen et al. [12] identified 20 carbonylated proteins in the matrix fraction of rice leaf mitochondria again mainly subunits of the respiratory chain, Krebs cycle enzymes, redox enzymes, or chaperones. In both studies it was concluded that the oxidation was not introduced by the isolation and separation procedure and therefore originated in the intact tissue. An in vitro oxidation of the rice matrix fraction caused an oxidation of additional 31 proteins belonging to the same general groups [12]. Sweetlove et al. [13] observed a decreased abundance of 12 mitochondrial proteins and breakdown products of 11 mitochondrial proteins in Arabidopsis cells subjected to oxidative stress, but whether this was a direct consequence of protein oxidation was not determined. In the present investigation we have identified proteins containing oxidized tryptophan in rice leaf and potato tuber mitochondria using two-dimensional gel electrophoresis followed by nano-HPLC-tandem mass spectrometry and bioinformatic analysis of the results.

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Materials and methods Rice (Oryza sativa L. ssp. japonicum, cv. Arborio) was grown as described [12] and potato tubers (Solanum tuberosum L. cv. Bintje) were bought in the local market. Intact and functional mitochondria were isolated from 8- to 10-day-old rice leaves and potato tubers using differential centrifugation followed by Percoll density gradient centrifugation. They were then subfractionated into submitochondrial particles (SMP) and matrix as described [12,14]. Proteins were separated by two-dimensional electrophoresis, either isoelectric focusing/PAGE [15,16] or by Blue-Native/PAGE [16 –18] and stained with Coomassie R-350. Protein spots were excised and digested [19] and analyzed by LC-MS/MS. A nanoflow capillary high-pressure liquid chromatography system (Famos, Switchos, Ultimate (LCPackings, The Netherlands)) equipped with a RP trap column (300 m i.d.  5 mm, PepMap C18, 5 m, LC-Packings) and a nanoscale RP capillary column (75 Am i.d.  15 cm, PepMap C18, 3 Am, LC-Packings) was set up for automated 2D-LC as described by the manufacturer. The nano-HPLC system was interfaced directly to a Q-TOF tandem mass spectrometer (Q-TOF Ultima Global, Micromass, UK) using the nano Z-spray electrospray source or to a Finnigan MAT LCQ ion-trap mass spectrometer using a Protana (Odense, Denmark) nanospray source equipped with 15 Am i.d. electrospray needles (New Objective). Peptides from each sample were separated by a 55 min gradient of 2 –50% (v/v) acetonitrile in 1% (v/v) acetic acid, 1% (v/v) formic acid. The flow rate in the second dimension was 200 –400 nl min 1. Both mass spectrometers employed data-dependent acquisition. The Q-TOF was calibrated using Glu-fibrinopeptide B (Sigma). Data analysis was performed using MassLynx and ProteinLynx software for Q-TOF and Excalibur software for the ion trap. The resulting MS/MS data set was analyzed using the Mascot search engine ver. 2.0.0 (Matrix Science, London, UK) on the NCBI nr database (December 2004). Tolerances for Mascot searches were different for data acquired from the two types of mass spectrometers. For the ion-trap MS tolerance was 1 Da, and 0.4 Da for MS for the Q-TOF. For both, MS/MS tolerance was 0.4 Da for matching in initial searches. Alignments of protein sequences was done using SIM (http://www.expasy.org/tools/sim.html). Results and discussion The soluble matrix fraction and the SMP containing mainly inner membrane were analyzed by 2D gel electrophoresis. The gels used were either IEF or BN in the first dimension and SDS-PAGE in the second. BN is particularly useful for the separation of membrane complexes such as the respiratory complexes [20]. A total of 195 protein spots, 169 from rice and 26 from potatoes, were analyzed and identified by MS/MS. Fig. 1 shows an IEF-PAGE 2D gel separation of matrix proteins from rice leaf mitochondria and the location of proteins found to contain N-formylkynurenine.

Fig. 1. Isoelectric focusing/SDS-PAGE two-dimensional gel of matrix proteins from rice leaf mitochondria. The spots noted in the text and in Table 1 are circled and numbered. 1 – 6, P protein of glycine decarboxylase; 7 – 9, aconitase; 10 – 11, methylmalonate semialdehyde dehydrogenase; 12 – 13, aldehyde dehydrogenase; 14 – 15, monodehydroascorbate reductase; 16 – 18, formate dehydrogenase; 19, superoxide dismutase. Spots 1, 7, 8, 13, 14, 16, and 17 did not contain N-formylkynurenine.

When the Mascot program identified a peptide as containing N-formylkynurenine with a significant score (or in a few cases a near significant score—see Table 1), the MS/MS fragmentation pattern was inspected manually to ascertain that the loss of the oxidized tryptophan from the peptide was in fact observed. An example is shown in Fig. 2. The doubly charged peptide of m/z 897.41 was found in the MS spectrum (not shown) corresponding to an addition of two oxygen atoms. In the MS/MS fragmentation the ion had a highly significant score. The spectrum shows a y-11 ion of 1234.63 and a y-10 ion of 1016.50, indicating a loss of 218.13 or the mass of tryptophan +32 mass units (Fig. 2). Using the above criteria of an increased peptide mass of 32 mass units, a significant peptide score and a fragmentation pattern showing loss of oxidized tryptophan, a total of 29 protein spots representing 17 different proteins were found to contain N-formylkynurenine (Table 1). With the exception of aconitase hydratase [citrate (isocitrate) hydrolyase; EC 4.2.1.3], all of the proteins were redox enzymes or subunits in protein complexes with a redox function such as glycine decarboxylase [EC 1.4.4.2, glycine dehydrogenase (decarboxylating)] and respiratory complexes I [EC 1.6.5.3, NADH dehydrogenase (ubiquinone)], III [EC 1.10.2.2, ubiquinol – cytochrome c reductase], and IV (EC 1.9.3.1, cytochrome c oxidase). Complexes I and III are known to be the major sites of ROS production in mitochondria [5,8] and damage may therefore have occurred close to the site of synthesis. Superoxide dismutase (SOD; superoxide:superoxide oxidoreductase; EC 1.15.1.1) interacts directly with superoxide (the substrate) and hydrogen peroxide (the product), so its oxidation is perhaps not too surprising. All but one [monodehydroascorbate reductase (NADH), EC 1.6.5.4] of the soluble rice proteins listed in Table 1 were also reported to be carbonylated by Kristensen et al. [12] using dinitrophenylhydrazine to tag the carbonylated proteins. This shows a very good correspondence between the

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Table 1 Oxidised tryptophan detected as N-formylkynurenine by mass spectrometric analysis in proteins from rice leaf and potato tuber mitochondria Protein

Rice leaves Putative glycine dehydrogenase [Oryza sativa (japonica cultivar-group)], P-protein

Aconitase [Lycopersicon pennellii] Methylmalonate-semialdehyde dehydrogenase [Oryza sativa] Aldehyde dehydrogenase [Oryza sativa] Putative monodehydroascorbate reductase [Oryza sativa (japonica cultivar-group)] Formate dehydrogenase, mitochondrial precursor (NAD-dependent formate dehydrogenase) (FDH) Probable superoxide dismutase (EC 1.15.1.1) (Mn) 2 precursor-rice Putative mitochondrial NADH:ubiquinone oxidoreductase 29 kDa subunit [Oryza sativa (japonica cultivar)] Putative mitochondrial processing peptidase, h [Oryza sativa (japonica cultivar-group)] Cytochrome c oxidase subunit 2 [Zea mays] Potato tubers Mitochondrial processing peptidase, a-subunit [Solanum tuberosum] Mitochondrial processing peptidase, h-subunit [Solanum tuberosum] NADH2 dehydrogenase (ubiquinone) (EC 1.6.5.3) chain 7-wheat mitochondrion 76 kDa mitochondrial complex I subunit [Solanum tuberosum] Unnamed protein product [Nicotiana plumbaginifolia] (superoxide dismutase)

Accession no.

Size of protein (kDa)

Coverage (%)

Sequence and position of oxidized peptide

Peptide score

Gel type/ Fraction/spot no.

GI|51090904

112

GI|33641855 GI|29027432 GI|2895866

112 99 58

GI|8163730

60

GI|50941197

53

24 31 12 15 29 9 47 37 55 58 27

982-EYAAFPAAwLR-992 982-EYAAFPAAwLR-992 982-EYAAFPAAwLR-992 982-EYAAFPAAwLR-992 984-EYAAFPAAwLR-994 67-IIDwENSAPK-76 23-STAAAASwLSDSASSPPR-40 23-STAAAASwLSDSASSPPR-40 117-KAFDEGPwPK-126 496-VTGwVNCFDVFDAAIPFGGYK-517 426-IATFwIDSDSR-436

49 50 64 49 47 68 96 94 55 44 54

IPG/MAT/2 IPG/MAT/3 IPG/MAT/4 IPG/MAT/5 IPG/MAT/6 IPG/MAT/9 IPG/MAT/10 IPG/MAT/11 IPG/MAT/12 IPG/MAT/12 IPG/MAT/15

GI|21263611

41

68

41 40

IPG/MAT/18 IPG/MAT/18

GI|7433347

25

66

GI|50908833

28

48

162-NFLPGYQQVVHGEwNVAGIAYR-183 283-GAIMDTQAVADACSSGQVAGYGGDV wFPQPAPK-315 125-LGwAIDEDFGSFEALVK-141 182-GANLVPLLGIDVwEHAYYLQYK-203 204-NVRPDYLSNIwK-215 123-CVLHAAwSAPTGLPADTLVDR-143 194-IPTGELwAGNPAR-206

68 48 65 48 69

IPG/MAT/19 IPG/MAT/19 IPG/MAT/19 IPG/MAT IPG/MAT

GI|55771316

54

24

36-SSGGFWTwLTGAR-48

53

IPG/SMP

GI|40795155

30

14

182-VTSADVLHSwAVPSLGVK-199

41

BN/SMP

GI|587566 GI|587564 GI|21493

60 59 55

GI|1084474

45

57 45 55 47 50 56

514-GPIQDLPDYNwFR-526 498-FIFDQDVAISALGPIQTLPDYNwFR-522 180-NPAFLDwEVK-189 180-NPAFLDwEVK-189 180-NPAFLDwEVK-189 226-GSGVCwDLR-234

80 51 56 51 58 59

IPG/SMP IPG/SMP IPG/SMP IPG/SMP IPG/SMP IPG/SMP

GI|758340

81

52

350-FQAVSwR-356

33

IPG/MAT

GI|19693

26

14 50

120-GSLGwAIDTNFGSLEALVQ-138 179-GANLVPLLGIDVwEHAYYLQYK-200

81 44

IPG/MAT IPG/MAT

The columns show the name and origin of the protein, the accession number, the size of the unprocessed protein, the percentage coverage of the full-length protein by the peptides identified (<20%-low, 20 – 40%-intermediate, 40 – 60%-high, >60%-very high), the sequence of the peptide containing N-formylkynurenine and its position in the protein, the score for the peptide (>44 is significant) and the 2D gel type (IPG – isoelectric focusing gel in the first dimension; BN – Blue Native gel in the first dimension), fraction (MAT – matrix, SMP – submitochondrial particles) and spot number for the spots shown in Fig. 1. ‘‘w’’ indicates oxidized tryptophan (N-formylkynurenine).

two methods possibly because the dinitrophenylhydrazinelabeling method can detect N-formylkynurenine [1]. Taylor et al. [11] also found N-formylkynurenine in aconitase and in subunits of the respiratory complexes I, III, and IV. Protein oxidation can lead to proteolytic breakdown of the modified protein molecule [10,21,22]. Sweetlove et al. [13] found a decreased amount and/or breakdown products of a number of proteins in mitochondria from cells exposed to oxidative stress. Their list included aconitase, methylmalonate-semialdehyde dehydrogenase [EC1.2.1.27, methylmalonate-semialdehyde dehydrogenase (acylating)], SOD, and the 75-kDa subunit of complex I, all found to contain oxidized tryptophan (Table 1).

The glycine decarboxylase complex (GDC) catalyzes together with serine hydroxymethyl transferase the oxidation of glycine as part of photorespiration in green leaves [23]. The complex is sensitive to oxidative stress through the modification of the lipoic acid residue by products of fatty acid peroxidation [24], but it is clearly also susceptible to Trp oxidation (Fig. 1, Table 1). The P protein of GDC was oxidized on Trp-990 (Trp-992 in spot 6) in a series of five spots on an IEF/PAGE 2D gel, which were identified with low to intermediate coverage (Table 1; Fig. 1). The spot with the most acidic pI in that necklace (spot 1) was also identified as P-GDC (21% coverage; not shown), but no oxidized Trp990 was detected. There is about 0.3 pH unit between the

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Fig. 2. Tandem mass spectrum of the doubly charged peptide of m/z 897.41 (Ser-23 to Arg-40) from methylmalonate semialdehyde dehydrogenase (Oryza sativa). This is from spot 10 in Fig. 1.

most acidic (spot 1) and the most basic (spot 5) spot. Spots 1 –5 are from one isoform of the P protein whereas spot 6 is from another, very similar, isoform with 97% identify. The predicted isoelectric points for accessions gi 551090904 and gi 33641855 are 6.35 and 6.51, which fits with their positions on the 2D gel. Spots 1 – 5 clearly represent the same protein with different posttranslational modifications (PTM) that change the isoelectric point significantly. One such PTM is protein phosphorylation, which is quite prevalent in plant mitochondria [25] and we have strong indications that the P protein of GDC is phosphorylated in vitro and possibly in vivo (B.K. Kristensen, N.V. Bykova, and I.M. Møller, unpublished observations). The P protein contains altogether

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six Trp residues (five in the C-terminus) out of which only two were found in identified peptides. The peptide containing unmodified Trp-997 was found in three of the spots whereas oxidized Trp-997 was never identified in any of the spots. This is opposite to the situation for Trp-990 (-992) (Table 1), indicating that Trp oxidation is not a random process and that certain Trp residues are particularly susceptible. Complex III catalyzes the transfer of electrons in the electron transport chain from ubiquinone to cytochrome c. In addition, the plant complex contains both the alpha and the beta forms of the mitochondrial processing peptidase (MPP; EC 3.4.24.64) as the core subunits [26]. Potato MPP containing N-formylkynurenine was identified in two series of spots (Table 1) with high coverage (45 –57%) while only one spot was found in rice with an intermediate coverage (24%) (Table 1). In potato, two different alpha forms showing an overall 72% amino acid identity contained oxidized Nformylkynurenine in a conserved position (Table 1, Fig. 3A). The presence of a lysine in position 502 in accession gi 587566 means that the peptide containing the oxidized tryptophan is much shorter than for accession gi 587564, which lacks the lysine and therefore a trypsin cleavage site (Fig. 3A). Both the alpha and beta forms of MPP were oxidized in potato. An alignment of the two showed 29% overall identity. The Trp-524 oxidized in the a-MPP was not conserved in h-MPP, while Trp-186 in h-MPP was not conserved in a-MPP (Fig. 3B). In rice only the beta form was oxidized and an alignment with potato h-MPP (58% identify) demonstrated that the site of oxidation differed, but not because of the absence of Trp residues. Both oxidized Trp-41 in rice and oxidized Trp-186 in potato h-MPP were conserved in the other species (Fig. 3C), but we cannot exclude the possibility that another oxidized Trp was overlooked in the rice protein due to the relatively low coverage. Mitochondrial Mn-SOD is responsible for removing the superoxide radical from the mitochondrial matrix (e.g., Ref. [8]).

Fig. 3. Conservation of the oxidized tryptophan residue in different forms of mitochondrial processing protease (MPP) of different origin. The stars below the two compared sequences indicate conserved amino acids.

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Rice leaf and potato tuber SOD showed 76% identity and all seven Trp were conserved (not shown). In rice SOD three of these, Trp-127, Trp-194, and Trp-214, were oxidized whereas Trp-107, Trp-156, and Trp-158 were found in unmodified peptides again pointing to a certain degree of selectivity. Similarly Trp-124 and Trp-191 in potato SOD (corresponding to Trp-127 and Trp-194 in rice SOD) were oxidized. SOD converts one ROS, superoxide, to another ROS, hydrogen peroxide, so the enzyme has presumably adapted to this interaction. At present we do not know how large a percentage of the SOD molecules contain modified tryptophan or indeed how this modification affects its function. In vitro oxidation of bacterial Mn – SOD causes an oxidation of a histidine residue in the active site [27]. A similar in vitro oxidation is seen for Cu,Zn – SOD, but this has not been detected in vivo possibly because the concentration of hydrogen peroxide is much lower in vivo than in the in vitro treatment [28]. It is tempting to speculate that the Trp residues act as intramolecular redox ‘‘decoys’’ diverting the ROS damage away from the active site and other sensitive domains in the protein. This could also be the case for other proteins. Aconitase catalyzes the interconversion of citrate and isocitrate in the Krebs cycle. This is not a redox reaction, but the enzyme has an Fe –S center in the active site, which is very sensitive to oxidation [29]. The enzyme was carbonylated in a yeast Mn – SOD knockout mutant [30]. Its amount was reduced and breakdown products were detected in mitochondria from oxidatively stressed Arabidopsis cells [13]. Finally, it contained oxidized tryptophan in human heart mitochondria [11] as well as in rice leaf mitochondria (Table 1). These are all indications of its sensitivity to oxidation. Two enzymes in mammalian cells and bacteria are capable of converting tryptophan into N-formylkynurenine, indoleamine-pyrrole 2,3-dioxygenase (EC 1.13.11.42), and tryptophan 2,3-dioxygenase (EC 1.13.11.11) and superoxide is involved in the reaction [1]. Neither of these enzymes has been identified in proteomic studies of plant mitochondria. In fact, neither has been reported to be present in plants. It is remarkable that virtually all of the proteins in plant mitochondria that contain N-formylkynurenine are either redox active themselves or subunits in redox-active complexes. To the first group belongs methylmalonate-semialdehyde dehydrogenase, aldehyde dehydrogenase (EC 1.2.1.-), MDA reductase, formate dehydrogenase (EC 1.2.1.2), SOD, and CCO subunit 2. To the second group belongs P-GDC, NADH-ubiquinone oxidoreductase (three subunits), and the a and h subunits of MPP. This is in contrast to the findings of Taylor et al. [11], who found N-formylkynurenine in several subunits of the ATPase complex, in the outer membrane porin, in a carrier protein as well as other proteins with no known direct connection to redox processes. Either ROS produced elsewhere caused the oxidation, or these proteins have a hitherto unrecognized redox connection. Acknowledgments We are grateful to Ina Blom for excellent technical assistance, to Helge Egsgaard for help with the mass spectrometer, and to

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