Lysine carbonylation is a previously unrecognized contributor to peroxidase activation of cytochrome c by chloramine-T

Ion mobility-assisted tandem MS uncovers hitherto overlooked modifications that are critical for the peroxidase activity of chloramine T-modified cytochrome c.


Introduction
Cytochrome c (cyt c) is a 12 kDa mitochondrial heme protein whose primary role is to serve as an electron shuttle in the respiratory chain. 1 Cyt c also possesses a second biologically relevant function as a peroxidase. [2][3][4][5][6][7][8] Peroxidases catalyze the oxidation (H abstraction) of substrates in the presence of H 2 O 2 . Catalytic turnover involves heme cycling between the Fe(III) and Fe(IV)]O states. 9 The peroxidase activity of cyt c is critical for triggering apoptosis (programed cell death) via oxidation of the mitochondrial membrane lipid cardiolipin. 7,10-12 Apoptosis is an essential process, e.g., for suppressing tumor growth. 13 Peroxidases require a pentacoordinated heme with a vacant distal coordination site where H 2 O 2 can bind, and where the Fe(IV)]O oxygen can be accommodated. 14 The heme in native cyt c is hexacoordinated, as the distal site is occupied by the Met80 sulfur (Fig. 1). 15 Dissociation of this Met80-Fe contact is a mandatory prerequisite for converting cyt c into a peroxidase. 14,16,17 There are different ways to achieve displacement of the distal Met80, such as unfolding, 14,16 chemical, 5,[17][18][19][20] or mutational modications, 4,7,18,[21][22][23][24][25][26] as well as cardiolipin binding. 3,[27][28][29] Exposure to basic pH induces the formation of "alkaline" conformations where Lys72, 73 or 79 serve as distal ligands instead of Met80. 20,[30][31][32][33][34] Chemical or mutational modications can shi this alkaline transition into the neutral range. 5,19,20,[35][36][37][38][39][40] We recently demonstrated 41 that H 2 O 2 converts native cyt c into an active peroxidase via a reaction sequence that starts with Tyr67 oxidation, followed by dissociation of the Met80-Fe contact. Subsequently, Lys72 or 73 occupy the distal coordination site. Carbonylation then abrogates the capability of Lys72/ 73 to serve as Fe ligands, thereby generating the free distal site that is required for an active peroxidase. 41 Lys carbonylation corresponds to the oxidation of the 3-amino group to an aldehyde (LysCHO, aminoadipic semialdehyde, DM ¼ À1 Da). 42,43 The most straightforward method to rupture the Met80-Fe bond is via Met oxidation to methionine sulfoxide (MetO). It is widely believed that this oxidative modication is sufficient for generating the open distal site that is required for peroxidase activation of cyt c in vitro and in vivo. 17,18,37,38,[44][45][46][47] In other words, this Met80-centric view envisions a simple causal relationship between MetO formation and peroxidase activity. 17,18,37,38,[45][46][47] A key pillar of the Met80-centric view comes from work on chloramine-T (CT) oxidized cyt c (CT-cyt c). CT is a mild oxidizing agent 48 that transforms into other reactive species in aqueous solution. 49 Among these, OCl À appears to be mainly responsible for cyt c oxidation, evident from the fact that cyt c exposure to CT and to OCl À causes similar effects. 17 CT-cyt c is a potent peroxidase. 17,37 Several studies have shown that CT-cyt c carries a sulfoxide at both Met80 and Met65, while claiming that no other chemical modications are present. 17,48,50 These claims go back to amino acid analyses conducted in the 1970s, which concluded that CT specically oxidizes Met, while leaving all other residues unmodied (except for -SH groups, which are absent in cyt c). 51 Those early studies 17,48,50,51 cemented the widely held belief that Met80 oxidation, without any other covalent changes, generates an active peroxidase. 17,18,37,[45][46][47] As a result, CT-cyt c has become a widely used model for peroxidase-activated cyt c. 17,37,38,47 Considering the far-reaching implications of work in this area (including the role of cyt c in apoptosis and tumorigenesis), 13 it is imperative to fully understand the properties of CTcyt c. Is it true that Met80 oxidation is sufficient for peroxidase activation? Is it true that MetO formation is the only CT-induced oxidative modication? Several lines of evidence hint at the involvement of additional factors. (1) Disruption of the Met80-Fe bond tends to produce "alkaline" ligation scenarios, where even at near-neutral pH the distal site becomes occupied by side chains such as Lys72/73. 19,35,36,38 These alternative ligation scenarios are promoted by the exible nature of the 71-85 U loop (Fig. 1). 52,53 Hence, it is not obvious why Met80 oxidation in itself would produce a vacant distal site that is required for peroxidase activity. (2) The purported strict selectivity of CT for Met is somewhat suspicious, considering that other oxidants can induce various modications at different types of residues. [54][55][56][57][58] (3) Typical CT-cyt c literature protocols utilize strong cation exchange (SCX) chromatography, which separates proteins according to their positive surface charge. Surprisingly, CT-cyt c undergoes SCX fractionation into several distinct bands. 37,38,47,48 MetO formation does not directly affect charge. The fact that CT-cyt c produces several SCX fractions thus suggests that the protein might contain additional, hitherto unidentied modications. The possible existence of such modications and their implications for peroxidase activation have been ignored thus far in the literature.
In this work we critically examined the structure and function of CT-cyt c. Various mass spectrometry (MS) techniques were applied to characterize CT-induced covalent modications in unprecedented detail. We were able to pinpoint previously unidentied features of the peroxidase activation process. Met80 oxidation alone was found to result in low peroxidase activity, because the distal site remained impeded by alkalinelike Lys ligation. Only aer opening of the distal site by CTinduced LysCHO formation did the protein show higher levels of activity. Previous studies on CT-cyt c that assumed a "clean" MetO protein have-in all likelihood-inadvertently characterized samples that contained both MetO and LysCHO sites. Our ndings argue against the prevailing notion that Met80 oxidation alone is sufficient for peroxidase activation. Instead, Lys carbonylation is a key peroxidase co-activator.

Preparation of CT-cyt c
Treatment of cyt c with CT was performed as described, 37 with minor alterations. Briey, a 5 mM aqueous solution of CT was added to 1 mM cyt c (both in 50 mM Tris buffer, pH 8.4) in a 1 : 1 volume ratio and reacted at room temperature for 3 hours. The reaction was then halted by dialysis against 10 mM potassium phosphate (pH 7.4) using 10 kDa MWCO lters (EMD Millipore, Etobicoke, ON). The CT-cyt c produced in this way was either used directly, or subjected to SCX chromatography using a 5 mL HiTrap sulfopropyl (SP) cartridge (GE Healthcare) on an AKTApurier FPLC. The HiTrap SP column was conditioned as per manufacturer instructions, and proteins were loaded at 0.25 mL min À1 . The column was then washed with H 2 O until the absorbance (280 nm) reached the pre-loading baseline. SCX elution was performed using a linear gradient of 500 mM ammonium acetate (pH 9) over 5 column volumes while collecting the eluent in 1 mL fractions.
Optical spectroscopy UV-Vis spectra were acquired on a Cary-100 spectrophotometer (Varian, Mississauga, ON). Soret measurement were performed on 10 mM protein solutions in 50 mM potassium phosphate buffer (pH 7.4). Absorbance measurements at 695 nm were performed at 40 mM protein. Peroxidase activity was measured as described 59 60 In these assays the formation of tetraguaiacol was probed by UV-Vis at 470 nm. 59 All activity assays contained 1 mM protein as determined by absorption measurements at the Soret maximum. Kinetic measurements at each H 2 O 2 concentration were repeated in triplicate. Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter (JASCO, Easton, MD), on 10 mM protein solutions in phosphate buffer (pH 7.4).

Mass spectrometry
MS experiments were performed on a Synapt G2 ESI quadrupoletime-of-ight instrument (Waters, Milford, MA) operated in resolution mode. The capillary, sample cone, and extraction cone voltages were set at +2.8 kV, 25 V, and 4 V, respectively. MS/MS experiments were performed using collision-induced dissociation (CID) with argon as collision gas. Protein samples for bottom-up MS experiments were prepared by overnight digestion using trypsin (50 : 1 protein/trypsin) at 37 C, and subsequently analyzed by liquid chromatography-mass spectrometry (LC/MS) using a nanoACQUITY UPLC (Waters) with a C18 column. Samples for intact protein MS were prepared as 10 mM solutions in a 50 : 50 solution of H 2 O : ACN with 0.1% formic acid, and directly infused at 5 mL min À1 using a syringe pump. For ion mobility-assisted top-down MS/MS experiments (top-down CID-IM-MS), 16+ protein ions were quadrupole-selected prior to CID in the trap collision cell. The collision energy used was 23 V for all samples. Ion mobility spectrometry (IMS) separation of CID products was performed in N 2 buffer gas at a wave velocity of 350 m s À1 and wave height of 13 V. Simulated isotope distributions were generated using ProteinProspector (UCSF). All simulated spectra correspond to the ferric (Fe(III)) state of cyt c.

Chloramine-T-induced Met80 oxidation
The CT-cyt c used in this study was prepared using established procedures. 37,38,47 An initial characterization was conducted by subjecting unfractionated samples to a standard tryptic digestion/LC-MS/MS workow. As expected, these assays revealed MetO formation (+16 Da) at Met80 and Met65, in line with earlier reports (Fig. S1 †). 17,37,46,47 No other +16 Da modications were detected. These observations appear to support the widely held belief that CT selectively oxidizes Met. 17,48,50,51 However, from the data discussed below it will be seen that standard mapping experiments (such as those of Fig. S1 †) provide an incomplete view of the CT-induced oxidation events.

SCX fractionation and optical characterization
SCX elution proles of CT-cyt c exhibited three major peaks ( Fig. 2A), consistent with previous observations. 37,38,47,48 Close examination of the highest intensity region revealed the presence of two partially overlapping signals (around 23 mL, equivalent to "peak B" in ref. 37). In total, therefore, SCX revealed the existence of four chromatographically distinguishable CT-cyt c forms. We will refer to these fractions as I, II, III, and IV in the order of decreasing retention. Fraction I had the largest retention volume, i.e., it interacted most strongly with the stationary phase, implying the most highly cationic character. The SCX behavior of fraction I was indistinguishable from that of unmodied cyt c. Conversely, fraction IV showed  Table S1 † for more information. Error bars represent standard deviations of three independent experiments. the weakest interaction with the stationary phase, i.e., proteins in this fraction carried the least amount of positive charge. From the A 280 values in the SCX chromatograms, the relative abundance of fractions IV, III, II, and I can be estimated to be roughly 10%, 35%, 35%, and 20%, respectively. For subsequent experiments we isolated thin slices out of the chromatograms for targeted experiments on each fraction ( Fig. 2A).
Optical spectra of the four CT-cyt c fractions were very similar to one another, but markedly different from those of unmodi-ed cyt c (Fig. S2 †). CT-cyt c showed a $30% CD intensity loss at 222 nm, revealing some decrease of a-helicity upon oxidation. 61 There was no concomitant increase in the random coil region ($200 nm), suggesting that CT-induced modications did not cause unfolding, but transitions to alternative folded structures. 61 A blue shi of the UV-vis Soret band from 409 nm to $405 nm indicated alterations in the heme environment. In addition, all CT-cyt c fractions exhibited loss of the 695 nm absorption band, signaling disruption of the Met80-Fe bond. 62 In summary, the spectroscopic data of Fig. S2 † indicate the formation of non-native, structured conformers in the CTtreated samples. The CD spectra were similar to those of Lysligated alkaline cyt c. 19,35,36 The blue-shied Soret band is reminiscent of alkaline cyt c as well. 34,38 Resonance Raman (RR) data in the literature showed fraction I to be Lys-ligated, whereas the remaining fractions were reported to have an open (OH À /H 2 O coordinated) distal site. 37 All these observations support the view that CT-cyt c shares structural features with previously characterized alkaline cyt c conformers. 19 Fraction I showed a relatively low activity; that of fraction II was twofold higher, and for fraction III an additional $twofold increase was observed. Fractions III and IV had similar activities. Under the low H 2 O 2 concentration conditions of Fig. 2B, turnover was barely detectable for unmodied cyt c. This activity trend can be summarized as follows: unmodied cyt c < fraction I < fraction II < fraction III z fraction IV. The same trend was observed when the experiments were repeated at a higher H 2 O 2 concentration of 500 mM, where even untreated cyt c showed non-zero activity (Fig. S3B †).
A number of earlier studies implied that CT treatment of cyt c generates a homogeneous product that is oxidized only at Met80 and Met65. 17,48,50 The data presented here demonstrate that this view is not correct. Instead, CT-cyt c is a mixture of at least four species that can be separated by SCX ( Fig. 2A) and that exhibit different peroxidase activities (Fig. 2B). While SCX fractionation of CT-cyt c has been demonstrated previously, 37,38,47,48 the chemical origin of this phenomenon and the associated activity differences remain unexplained. SCX separates proteins according to their cationic surface charge. CTinduced Met / MetO conversion does not directly alter the ionic nature of the protein (Fig. S4 †). Conformational factors could, in principle, contribute to the SCX fractionation. 48 However, the nearly indistinguishable CD and UV-Vis spectra point to similar overall structures for fractions I-IV (Fig. S2 †), making conformational factors an unlikely culprit for the observed SCX behavior.

SCX fractions represent specic proteoforms
For uncovering the origin of the SCX properties and activity differences of fractions I-IV we applied a range of MS methods. As an initial step, intact protein mass spectra were acquired (Fig. 3). At rst glance, the mass distributions of the four fractions appear to be almost identical, reminiscent of the optical data in Fig. S2. † Each spectrum in Fig. 3 is dominated by two signals that are spaced +16 Da apart, as expected for MetO formation at Met80 and/or Met65. In addition, each fraction showed a low intensity signal close to the mass of unmodied cyt c. In Fig. 3 these signals were annotated according to the number (0, 1, 2) of MetO formation events. This interpretation reects the existing literature, which assumes that CT specically targets Met80 and Met65 while leaving other residues unmodied. 17,37,38,47,48,50,51 To test the viability of this simple MetO-centric interpretation we calculated theoretical isotope distributions for unmodied cyt c, a +16 Da variant (one MetO), and a +32 Da variant (two MetO). These theoretical distributions were superimposed on the experimental spectra (Fig. 3, top row). Fraction I showed excellent agreement with the theoretical data. In contrast, fractions II, III, and IV exhibited gradually increasing mass defects, i.e., the measured peaks had a lower mass than expected. The magnitude of this discrepancy correlated with the SCX elution behavior: Fraction IV showed the largest discrepancy, while fraction I exhibited no discrepancy. The mismatch between experimental spectra and the theoretical "clean" +16 Da adducts implies that MetO formation is not the only type of covalent modication in fractions II-IV. Control experiments conrmed that mass discrepancies were already observable for unfractionated CT-cyt c, i.e., the observed effects were not an SCX-related artifact (Fig. S5 †).
The missing piece of the puzzle that explains the properties of CT-cyt c is Lys carbonylation (Fig. S4 †). This reaction is known to be facilitated by metal centers (i.e., heme in cyt c). 43 It involves a radical intermediate 63 that gets converted to 3-hydroxylysine. Elimination of ammonia then produces LysCHO. 64 Each LysCHO formation event is accompanied by a À1 Da shi. 41,64 Compilations of known oxidative protein modications reveals LysCHO formation to be the only process associated with a À1 Da shi. 42,43 The direct MS-based detection of LysCHO sites will be discussed below. Returning to Fig. 3, revised modeling of the mass spectra to account for a combination of MetO (+16 Da) and LysCHO formation (À1 Da) resulted in excellent agreement between experimental and simulated spectra for all fractions (Fig. 3, bottom row). The isotope models suggest that fractions I, II, III, and IV carry 0, 1, 2, and 3 LysCHO groups, respectively. These modications are in addition to MetO formation at Met80 and Met65. 17,37,38,47,48,50,51 The different numbers of LysCHO groups provide an explanation for the SCX behavior of the four fractions. The SCX strategy used here separates proteins on the basis of their overall cationic charge; each LysCHO site reduces this charge by one unit. 41,42,64 In fraction I, the lack of Lys modications causes the most positive surface charge, similar to that of unmodied cyt c (pI 9.6). 65 Hence, fraction I (and unmodied cyt c) interact most tightly with the SCX column, resulting in the largest elution volume (Fig. 2A). On the other extreme, fraction IV with its three LysCHO has lost three positive charges, causing those proteins to interact most weakly with the column. The behavior of fractions II and III falls in-between these two extremes.
In summary, LysCHO formation accounts for the previously unexplained observation 37,38,47,48 that CT-cyt c can be fractionated by SCX. LysCHO has been shown to play a role in the peroxidase activation of cyt c by H 2 O 2 . 41 However, LysCHO formation in cyt c has not been reported for oxidizing agents other than H 2 O 2 . The number of LysCHO sites is the only detectable difference in the covalent structures of fractions I-IV, implying that LysCHO formation must be responsible for their different activities. Combining the information of Fig. 1B and 3, the peroxidase activity can be ranked according to the number of LysCHO: fraction I (0 LysCHO) < fraction II (1 LysCHO) < fraction III (2 LysCHO) z fraction IV (3 LysCHO).

Top-down CID-IM-MS for proteoform-selective analysis
Mapping of LysCHO sites poses signicant analytical challenges. 66 Typical bottom-up MS assays struggle with this task due to LysCHO-induced interference with tryptic cleavage and losses of ionization/fragmentation efficiency. 42,67,68 Thus, it is not surprising that previous MS investigations failed to detect LysCHO in CT-cyt c. 17,18,37,38,[45][46][47][48]50,51 To address these challenges we applied top-down MS/MS, i.e., gas phase fragmentation of undigested proteins. 69 Initial attempts were unsuccessful due to the complexity of the fragment ion spectra. This problem was solved by incorporating ion mobility (IM) spectrometry to provide an additional separation dimension, thereby implementing a "top-down CID-IM-MS" workow ( Fig. S6 †). Similar experiments have previously been applied to simple model systems, [70][71][72] but this work marks the rst time that such an approach was used for tracking unknown biologically relevant modications.
For validation, top-down CID-IM-MS was initially applied to peak 1 of fraction I (which is devoid of LysCHO) to examine the initial MetO formation event (Fig. 4). All C-terminal fragments starting at y 25 showed a +16 Da shi relative to unmodied cyt c. The y 25 to y 24 transition allowed the oxidation site to be iden-tied as Met80 with single-residue resolution. The presence of other oxidation sites in peak 1 (including Met65) could be ruled out. Measurements on peak 2 (+32 Da) veried Met80 and Met65 as the only two oxidation sites in fraction I (Fig. S8 †). Taken together, these data imply that CT-induced MetO formation is a sequential process, with Met80 as the initial oxidation target whereas Met65 is affected at a later stage.

Mapping of LysCHO sites
We next sought to identify the locations of LysCHO groups in fractions II, III, and IV by top-down CID-IM-MS. According to Fig. 3, these fractions possess 1, 2, and 3 carbonylation sites, respectively. LysCHO sites can be identied from À1 Da mass shis of fragment ions. The experiments were facilitated by the fact that, fortuitously, peak 1 in all fractions corresponded exclusively to MetO formation at Met80 (Fig. S9 †). Thus, LysCHO sites in the various fractions could be mapped via comparisons with fraction I fragments. By using this internal reference we could unequivocally rule out calibration errors as an explanation for the observed À1 Da shis. Our assignments were further veried by simulated isotope distributions that were overlaid onto the experimental spectra. Fig. 5 reveals that for all fractions, N-terminal fragments up to b 46 , as well as C-terminal fragments up to y 29 were indistinguishable from those of fraction I. This nding connes all LysCHO sites to residues 47-75 which comprise ve out of the total 19 Lys residues. In fraction II, y-ions from y 38 to y 58 showed a À1 Da shi. The y 38 to y 29 transition narrowed down the location of this modication to 67 YLENPKK YI 75 . Hence, the single LysCHO site in fraction II is located on Lys72 or Lys73 (Fig. 5, second row).
Fraction III also showed a y 38 to y 29 À1 Da transition, implying one LysCHO modication on Lys72/73. An additional transition took place between y 40 and y 58 , localizing the second LysCHO site to the region 49 TDANKNKGITWKEETLM 65 . Thus, the second modication site in fraction III is located on Lys53, Lys55, or Lys60 (Fig. 5, third row).
Pinpointing the three LysCHO sites in Fraction IV was complicated by diminished fragmentation efficiencies that translated into low S/N ratios. This phenomenon likely reects the loss of protonation sites aer LysCHO formation, keeping in mind that the proximity to H + sites favors b/y ion formation. 73-75 y 58 exhibited a mass shi of À3 Da (Fig. 5, bottom row), con-rming the presence of three LysCHO sites, as inferred from Fig. 3. From the fragment ion mass shis in Fig. 5 (bottom row), the predominant modication pattern of fraction IV appears to be a single LysCHO in the Lys53/55/60 range, plus two LysCHO at Lys72/73. However, we cannot rule out that the nal LysCHO formation event also affects Lys53/55/60 to some extent. The similar peroxidase activities of fractions III and IV suggest that this third LysCHO is of minor functional importance. Experiments analogous to those of Fig. 5 were conducted on peak 2 of each fraction, yielding LysCHO patterns similar to those of peak 1 ( Fig. S10 and S11 †). Fig. 5 implies that CT-mediated LysCHO formation is a wellordered process. Starting from LysCHO-free protein (fraction I), carbonylation initially affects Lys72/73 (fraction II). The next carbonylation event takes place in the Lys53/55/60 region, generating fraction III. The nal LysCHO site may affect Lys72/ 73 or Lys53/55/60. As noted above, MetO formation is also a sequential process, initially affecting Met80 and subsequently Met65.

LC-MS/MS peptide mapping revisited
The detection of LysCHO sites by top-down CID-IM-MS may seem at odds with previous studies on CT-cyt c that only found MetO formation. 17,37,38,47 In intact protein MS data the small (À1 Da) mass shis associated with LysCHO formation are easily overlooked, unless the spectra are carefully scrutinized via isotope modeling (Fig. 3). Similarly, LysCHO detection by traditional tryptic digestion LC-MS/MS (Fig. S1 †) is difficult due to reduced fragmentation resulting from charge site loss, and suppression of tryptic cleavage. 42,[73][74][75] These issues are exacerbated for the situation encountered here, where up to three Fig. 4 Top-down CID-IM-MS data that pinpoint MetO formation in fraction I. The sequence is shown along the top, with key fragments indicated. Bottom: selected fragment ions from an unmodified control (red), and from peak 1 of fraction I (blue, see Fig. 3). Lines/dots represent theoretical isotope distributions calculated for unmodified fragments ("no ox."), or for MetO containing fragments. Met80 is the only covalent modification in peak 1 of fraction I.
Armed with the information from top-down CID-IM-MS, we revisited the tryptic digestion LC-MS/MS workow with careful optimization of experimental parameters. Gratifyingly, we were able to conrm carbonylation of Lys72 in the missed-cleavage peptide T13-14 (residues 61-73, Fig. 6). Consistent with the data reported above, this LysCHO peptide was found in fractions II-IV, but not in fraction I. No other LysCHO sites were detected by LC-MS/MS. Thus, the top-down CID-IM-MS work-ow is superior for this type of application, as it allowed the detection of all three carbonylation sites (Fig. 5).

Mechanism of CT-induced peroxidase activation
The data of this work provide a clear view of the sequential CTinduced events that convert cyt c into an active peroxidase. This mechanism, summarized in Fig. 7, is governed by the undisputed fact that peroxidase activity requires the distal coordination site to be accessible. 14,16,17 Unmodied (native) cyt c has the lowest peroxidase activity of all the forms studied here because its distal ligation site is blocked by Met80. Very slow substrate turnover under these conditions may take place as the result of conformational uctuations 52,53 that occasionally disrupt the Met80-Fe bond. 40,41,76 CT-induced oxidation of Met80 generates fraction I, an "alkaline" conformer where Lys72 or Lys73 has moved into the ligation site previously occupied by Met80. This ligation switch is supported by RR data 37 as well as NMR and EPR experiments. 38 While the enhanced peroxidase activity of alkaline Lys-Fe conformers is well documented, 38 the origin of this effect is somewhat unclear because transient opening of the Lys-Fe contact takes place more slowly than for Met-Fe. 34,77 Likely, the enhanced activity of fraction I is not caused by hexacoordinated Lys-Fe species per se, but by pentacoordinated structures that are part of the fraction I ensemble. 14,38 RR spectra are consistent with this interpretation, suggesting the presence of $15% pentacoordinated protein in the predominantly Lys-Fe coordinated fraction I ("peak A" in Fig. S13 of ref. 37). Other factors may contribute to the peroxidase activity of Lys-ligated cyt c as well, 41 one of these is the increased heme pocket volume which facilitates substrate access. 19,77 As the next step in Fig. 7, carbonylation abrogates the iron ligating capability of Lys72/73, thereby generating fraction II. Fraction II still does not represent a genuine pentacoordinated conformer, evident from the fact that subsequent Lys53/55 carbonylation further enhances peroxidase activity (our MS data suggested the possible participation of Lys60, but we exclude this residue for reasons outlined below). Our data are thus consistent with a scenario where Lys53/55 participate in iron ligation in fraction II. However, these Lys53/55-Fe contacts must be quite labile, evident from the fact that fraction II has a higher activity than both fraction I and unmodied cyt c (Fig. 2B).
Carbonylation of Lys53/55 subsequently produces fraction III which possesses a permanently open distal site and shows the highest peroxidase activity. RR data conrm that fraction III is pentacoordinated, as expected for a functional peroxidase. 37 A nal carbonylation event produces fraction IV which retains both an open distal site and peroxidase activity.
The essential point in the activation cascade of Fig. 7 is that CT-induced Met80 oxidation only results in a moderate level of peroxidase activity, because the distal site remains congested by Lys ligation. Subsequent CT-induced LysCHO formation frees up the distal site, thereby producing a genuine pentacoordinated heme. Previous work attributed the peroxidase activity of CT-cyt c exclusively to MetO formation, 17,18,37,38,[44][45][46][47] failing to recognize the occurrence and mechanistic signicance of Lys carbonylation. Our data reveal that MetO formation only represents an initial step en route toward peroxidase-activated CT-cyt c. Lys carbonylation represents a co-activator that boosts the activity by a factor of $4 (fractions III/IV) relative to the MetO-only form (fraction I, see Fig. 2).
The model of Fig. 7 provides a simple explanation for the correlation between the activity of the CT-cyt c fractions and their SCX behavior: both factors are directly tied to the number of LysCHO sites. When accounting for the SCX fractionation behavior, one has to remember that while the protein interacts with the anionic stationary phase, it may adopt structures and ligation scenarios that differ from the bulk solution conformations of Fig. 7. 78 The ability of cyt c to adopt various distal ligation scenarios is consistent with the foldon model that dissects the protein into regions of different stability (Fig. S12 †). 52 The gray and red foldons (40-57 and 71-85 U loops, respectively) show the highest exibility. 52 Not surprisingly, the alternative Fe ligands identi-ed above are located in these two exible regions, i.e., Lys72/73 (red) and Lys53/55 (gray). Opening of gray 79 and red 53 has previously been linked to alkaline conformers. The exibility of gray and red also governs the CT-induced transitions outlined in Fig. 7. In native cyt c the red foldon is anchored by the Met80-Fe linkage. 52,53,79 Our data suggest that MetO formation sterically perturbs this (already labile) 52 region, thereby triggering rearrangements that allow Lys72/73 to move into the distal site. 53 The foldon hierarchy dictates that red can only open up when gray is already unfolded. 52,53,79 Hence, aer carbonylation of Lys72/73, the plasticity of the unfolded gray foldon will allow Lys53/55 to move into the vacated distal site. Our MS data suggest that Lys60 could also participate in Fe ligation, but its placement in the relatively rigid yellow foldon renders such a scenario less likely (Fig. S12 †).
Our data do not provide evidence for carbonylation of Lys79. In principle, this residue represents another possible ligand that might block access to the iron aer Met80 oxidation. 38 However, evidence for such Lys79-Fe contacts comes primarily from work on K72A yeast iso-1 cyt c which has coordination propensities that are different from those of the horse wild type studied here. 38 Our ndings suggest that Lys79-Fe contacts do not play a major role in Met80-oxidized horse cyt c, such that peroxidase activation is attainable without Lys79 carbonylation.

Conclusions
Our discovery of multiple, previously unreported carbonylation events in CT-cyt c calls into question the prevailing Met80centric view of peroxidase activation, according to which oxidation of Met80 alone is sufficient for turning cyt c into a pentacoordinated peroxidase. 17,18,37,38,44,45,47,48 The conclusions from a number of those reports have relied on the alleged delity of CT for specic Met oxidation. Several studies pooled the highest intensity region in the SCX chromatogram, assuming that samples obtained in this way represent the expected "clean" MetO species. 37,38,47,48 Our data strongly suggest that such samples correspond to fractions II/III, with oxidation at Met80, Met65, and with LysCHO sites in the Lys72/73 and Lys53/55 range. Carbonylation of these samples enhances their peroxidase activity almost fourfold relative to fraction I, which represents the true MetO-only species (Fig. 2 and 7). It appears that past studies inadvertently studied carbonylated CT-cyt c proteoforms, misattributing some of the carbonylation-induced effects to MetO formation.
Previous studies on CT-cyt c yielded conicting results, suggesting that the distal site is open 37 or occupied by Lys. 38 The present work helps resolve these discrepancies. CT-cyt c comprises multiple species with different coordination patterns (Fig. 7). Thus, any results will depend on the precise experimental conditions used for isolating protein from the CT-cyt c reaction mixture.
Only four out of the 19 Lys residues in cyt c were identied as possible carbonylation sites. In native cyt c these four residues are relatively close to the distal face of the heme, which represents the site of peroxidase catalysis (Fig. 1). Considering that all 19 Lys residues are solvent-exposed (Fig. S13 †), the spatial selectivity of LysCHO formation rules out diffusion-mediated oxidation events, where modications are governed by solvent accessibility. 55 It instead appears that LysCHO formation is heme-dependent. 43,58 This line of thought bolsters our conclusions, as carbonylation of Lys53/55/72/73 conrms the capability of these residues to come in direct contact with the heme by serving as alternative Fe ligands (Fig. 7).
There are certain parallels, but also differences between the CT-induced peroxidase activation of cyt c studied here, and the H 2 O 2 -induced activation that we explored earlier. 41 The initial transformation triggered by H 2 O 2 is oxidation of Tyr67, while this residue remains unmodied during CT exposure. Peroxidase activation by both CT and H 2 O 2 involves sequential modications and transiently populated alkaline conformers, ultimately producing a vacant distal site. One key difference is that CTinduced activation relies on MetO formation for disruption of the Met80-Fe bond (Fig. 7). In contrast, H 2 O 2 causes this bond to break as a result of Tyr67 oxidation, without Met80 oxidation. The reasons underlying the different initial reactivities of the two oxidants remain to be fully explored. Likely, H 2 O 2 oxidation of Tyr67 proceeds with catalysis by the iron center, 43 whereas CT can oxidize Met without heme involvement. 51 A common feature of CT and H 2 O 2 -induced activation is LysCHO formation which prevents the distal site from being blocked by Lys side chains. It is likely that LysCHO formation plays a similar role for the conversion of cyt c into an apoptotic peroxidase under other conditions, e.g., aer cardiolipin binding 3,27-29 in vitro and in vivo. However, this hypothesis remains to be veried experimentally.

Conflicts of interest
There are no conicts to declare.