Meena
Kathiresan
and
Ann M.
English
*
Concordia University Faculty of Arts and Science, and PROTEO, Chemistry and Biochemistry, Montreal, Canada. E-mail: ann.english@concordia.ca
First published on 7th September 2016
We recently reported that cytochrome c peroxidase (Ccp1) functions as a H2O2 sensor protein when H2O2 levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (CycII), determines whether Ccp1 acts as a H2O2 sensor or peroxidase. For H2O2 to serve as a signal it must modify its receptor so we employed high-performance LC-MS/MS to investigate in detail the oxidation of Ccp1 by 1, 5 and 10 M eq. of H2O2 in the absence of CycII to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H2O2 at Ccp1's heme. This results in the incorporation of ∼20 oxygen atoms predominantly at methionine and tryptophan residues. Extensive intramolecular dityrosine crosslinking involving neighboring residues was uncovered by LC-MS/MS sequencing of the crosslinked peptides. The proximal heme ligand, H175, is converted to oxo-histidine, which labilizes the heme but irreversible heme oxidation is avoided by hole hopping to the polypeptide until oxidation of the catalytic distal H52 in Ccp1 treated with 10 M eq. of H2O2 shuts down heterolytic cleavage of H2O2 at the heme. Mapping of the 24 oxidized residues in Ccp1 reveals that hole hopping from the heme is directed to three polypeptide zones rich in redox-active residues. This unprecedented analysis unveils the remarkable capacity of a polypeptide to direct hole hopping away from its active site, consistent with heme labilization being a key outcome of Ccp1-mediated H2O2 signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O2 reactivity.
Within the heme peroxidase class of oxidoreductases, there is a dramatic variation in the number of oxidizable residues present in their polypeptides.7 For example, manganese peroxidase possesses just one tryptophan and no tyrosine, whereas cytochrome c peroxidase (Ccp1) is studded with redox-active aromatic residues (Fig. 1). Thus, Ccp1 is endowed with a high capacity for hole hopping within its polypeptide, which must be pertinent to its physiological function. In vitro, Ccp1 efficiently couples the two-electron reduction of H2O2 to the one-electron oxidation of two ferrocytochrome c (CycII) molecules:8
Ccp1III + H2O2 → CpdI(FeIV, W191˙+) + H2O | (1) |
CpdI(FeIV, W191˙+) + CycII → CpdII(FeIV) + CycIII | (2) |
CpdII(FeIV) + CycII + 2H+→ Ccp1III + CycIII + H2O | (3) |
Fig. 1 Oxidizable residues in Ccp1. PyMOL-generated cartoon of Ccp1 (PDB 1ZBY) showing the protein's 14 tyrosines (Y, green), 7 tryptophans (W, blue), 6 histidines (H, orange), 5 methionines (M, grey) and the single cysteine (C, magenta). Solvent-exposed residues are underlined. |
Ccp1III is the resting ferric enzyme and compound I (CpdI) and compound II (CpdII) are catalytic, high-valent oxyferryl (FeIV) intermediates. The second of the two oxidizing equivalents (i.e., the two electron holes) in CpdI is localized on W191. This residue forms a stable cationic radical, which was the first tryptophanyl radical identified in a protein.9,10 Notably, W191 lies in the electron-transfer pathway between the Ccp1 and Cyc hemes and mutation of this residue to non-redox active phenylalanine gives the W191F variant, which exhibits negligible CycII-oxidizing ability.11
Ccp1's catalytic cycle has been examined in exquisite detail in vitro over several decades as a model of heme-peroxidase catalysis.8 Despite the intense focus on Ccp1 as a peroxidase, we have recently reported that it mainly functions as an H2O2 sensor protein in yeast mitochondria.12,13 As yeast cells switch from fermentation to respiration, heme-free apoCcp1 escapes from the mitochondria.12,14 Concomitantly, the activity of the peroxisomal–mitochondrial catalase (Cta1) increases and we have gathered strong evidence that apoCta1 is a recipient of Ccp1's heme.14 Intracellular H2O2 levels spike as cells begin to respire,12,14 and the proximal heme ligand, H175 (Fig. 1), is extensively oxidized in Ccp1 isolated from respiring yeast.14 Thus, labilization of Ccp1's heme on H175 oxidation enables its transfer to apoCta1, converting the latter into a powerful catalytic H2O2 scavenger.
To better understand this unprecedented mechanism of H2O2-regulated heme transfer, detailed characterization of Ccp1 modification by excess H2O2 in the absence of substrate was undertaken. Heme-mediated reduction of H2O2 by endogenous donors in Ccp1 in the absence of CycII is well-documented in vitro15 but the oxidized forms of Ccp1 have been poorly characterized. Repeated two-electron reduction of H2O2 by Ccp1 requires repeated intramolecular radical transfer or hole hopping from the oxidized heme. This will generate new transient radicals in the polypeptide, which will hop to new sites before being trapped as stable oxidation products (eqn (5)) or the nascent radicals may be trapped (eqn (6)):15
Ccp1III + H2O2 → CpdI + H2O | (4) |
CpdI → Ccp1III(A˙, B˙) → Ccp1III(Cox, Dox) | (5) |
Ccp1III(Cox, Dox) + H2O2 → →Ccp1III(Cox, Dox, Eox, Fox) + H2O | (6) |
Ccp1's ability to endogenously reduce up to 10 M eq. of H2O2 stems from its abundance of oxidizable residues (Fig. 1).7 Polypeptide oxidation has been confirmed by the detection of transient radicals in CpdI and overoxidized Ccp1 (defined here as Ccp1 oxidized by >1 M eq. of H2O2) by spectroscopic studies on the wild-type protein and its variants.16–21 A broad EPR signal at 4 K was unequivocally assigned to W191˙+ and a narrow EPR signal to Y71˙ and Y236˙.16–21 Trapping of radicals provides additional evidence for oxidation of Ccp1 at multiple residues. Spin adducts can be characterized by mass spectrometry (MS),22–26 and Y˙ radicals trapped in Ccp1 by 2-methyl-2-nitrosopropane (MNP) give MNP mass adducts that were localized to tyrosine-containing tryptic peptides T6 (Y36, Y39, Y42) and T26 (Y229, Y236),23 as well as to specific residues, Y39, Y236 and Y153.22 Efficient radical quenching by TEMPO˙ (2,2,6,6-tetramethylpiperidinyl-1-oxy) also generates mass adducts amenable to MS analysis.26 We isolated several TEMPO-labeled peptides from digests of overoxidized Ccp1, including T6, T14 + T15 (W126), T18 + T19 (Y153), T23 (Y187, W191, Y203, W211), T27 + 28 (Y244, Y251) and T28 (Y251), where the oxidizable residues are in brackets, but the actual residue(s) labeled in each peptide was (were) not identified.26
EPR investigations detect the more stable radicals in proteins, notably those with low O2-reactivity, as the results of this study suggest. Spin trapping and scavenging can identify less stable radicals but those that react with spin traps and scavengers tend to be exposed on a protein's surface because of steric hindrance. We rationalized that high-performance MS, the current method of choice for the qualitative and semiquantitative characterization of oxidative protein modification,27–30 would allow us to identify all residues in Ccp1 that serve as endogenous donors, including those that undergo only small mass changes on oxidation. Indeed, the LC-MS/MS results described here provide a comprehensive map of the residues modified on heme-mediated oxidation of Ccp1 by 1 M eq. of H2O2, which generates CpdI (eqn (4)),15 and by 5 and 10 M eq. of H2O2. Multiple cycling of the heme back to its ferric form by hole transfer to the polypeptide (eqn (5) and (6)) enables repeated H2O2 activation and reduction at the heme iron, leading to a highly overoxidized protein.15,22,23,31–35
The chemical nature of the stable oxidation products and their location within Ccp1's polypeptide are identified by LC-MS/MS. The results are interpreted by considering both the intrinsic reactivity of the amino acid radicals formed on hole hopping from the heme as well as their proximity to conserved internal waters and to regions of O2 density found by molecular dynamics (MD) simulations. Overall, our results reveal that extensive H2O2-initiated hole hopping can be accommodated in a seemingly controlled manner within a relatively small protein matrix. We also elucidate how heme-mediated oxidation of Ccp1 supports its remarkable role in yeast as a H2O2 sensor and signaling molecule that partakes in H2O2-regulated heme transfer. Until recently H2O2 was viewed as a toxic by-product of aerobic metabolism and associated with many pathologies and biological aging.36–39 However, H2O2 signaling is now known to mediate many physiological processes via thiol- and metal-catalyzed protein oxidation.40,41 Furthermore, our study suggests that extensive protein oxidation may be physiological and not just pathophysiological.
(7) |
The numerator sums the normalized PAs of all peptides containing Xox (PAox) and the denominator sums the normalized PAs of all peptides containing any form of X. The relative standard deviation of the reference peptide PAs is ∼4% (Table S2†), which reflects the precision in the percent oxidation reported here.
R˙a | pKa of R˙+ | Reduction potential E7 of R˙+ at pH 7 (V) | Peroxy radical reported | k (M−1 s−1) for R˙ + O2 reaction |
---|---|---|---|---|
a R˙ is the neutral amino acid radical of tyrosine (Y), tryptophan (W), cysteine (C), histidine (H) and methionine (M). b Note that k = 5 × 108 M−1 s−1 for dimerization of Y˙ to dityrosine.64 c NR, not reported. | ||||
Y˙b | −2 (ref. 49) | 0.93 (ref. 50 and 51) | Yes (ref. 52) | <103 (ref. 53) |
W˙ | 4 (ref. 50) | 1.01 (ref. 50, 51 and 54) | Yes (ref. 55) | <106 (ref. 56) |
C˙ | NRc | 0.92 (ref. 57) | Yes (ref. 58 and 59) | 6.1 × 107 (ref. 60) |
H˙ | 5–7 | 1.17 (ref. 61) | Yes (ref. 62) | NR |
M˙ | −6 (ref. 63) | 1.5 (ref. 63) | NR | NR |
Fig. 2 Deconvolved mass spectra showing that Ccp1 oxidation by H2O2 is mediated by its heme. Oxidized Ccp1 (1 μM) was diluted 5-fold into the MS solvent and 5 μL aliquots were analyzed by LC-MS on a Waters QToF3 mass spectrometer. Mass spectra of (A–C) holoCcp1 oxidized with 0, 1 and 10 M eq. of H2O2 and (D) apoCcp1 oxidized with 10 M eq. of H2O2. The observed mass of the unoxidized polypeptide is 33730.50 ± 1.35 u (calc. 33730.33 u) and overoxidation of holoCcp1 gives incremental mass shifts of +16 u, which are not observed for apoCcp1 (panels B and C vs. D). Experimental details are provided in the ESI.† |
Fig. 3 Methionine and cysteine oxidation. Ccp1 (1 μM) in KPi/DTPA was treated with the indicated molar ratio of H2O2 for 1 h at room temperature, digested with trypsin and the peptides were analyzed by LC-MS/MS as described in the ESI.† Percent (A) methionine oxidation to MetO (+16 u); (B) C128 oxidation to CysSO2H (+32 u) and CysSO3H (+48 u). Yields are based on peptide PAs (eqn (7)) from three independent experiments (n = 3) and presented as averages ± SD. Solvent-exposed methionines are underlined in panel A. |
A single buried cysteine residue (C128) is located >20 Å from the heme in the distal domain. In CpdI ∼3% of C128 is oxidized to CysSO2H/CysSO3H, and the oxidized forms sum to 60% and 100% on treatment with 5 and 10 M eq. of H2O2, respectively (Fig. 3B), revealing that C128 also acts as a donor to the heme.
Ccp1's seven tryptophans undergo extensive H2O2-induced hydroxylation and up to 15% of W223 is additionally converted to kynurenine (Fig. 4). Notably, W191, W211, W223 proximal to the heme are ∼5–40% oxidized by 1 M eq. of H2O2, whereas the distal W57 and W126 are extensively oxidized by 5 M eq. of H2O2 but W51 at 3.1 Å from the heme is modified only in protein exposed to 10 M eq. of H2O2 (Fig. 4). Furthermore, oxidized W51 and W57 are detected solely as Trp(OH)2 (dihydroxytryptophan), from which we infer that their TrpOH form is readily oxidized as hole hopping to the distal domain increases in overoxidized Ccp1. W101, located on Ccp1's distal surface at >25 Å from the heme, undergoes little oxidation (Fig. 4) probably because hole hopping to this residue is blocked by O2 scavenging of radicals on W126 or C128 or other residues closer to the heme (Fig. 1).
Fig. 4 Tryptophan residues undergo extensive mono- and dihydroxylation. Percent tryptophan oxidation to (A) TrpOH, (B) Trp(OH)2 and kynurenine (inset). Experimental details are given in the caption to Fig. 3. Solvent-exposed tryptophans are underlined. |
Fig. 5 Tyrosine oxidation products include TyrOH and dityrosine. Percent tyrosine oxidation to (A) TyrOH (+16 u) and (B) dityrosine (−2 u) in T6 (Y36, Y39, Y42), T8 (Y67, Y71) and T26 (Y229, Y236). Experimental details are given in the caption to Fig. 3. Solvent-exposed tyrosines are underlined in panel A. |
The doubly and triply charged ions of peptides T6 and T8 from untreated Ccp1 show high intensity MS1 signals (data not shown). Overoxidized Ccp1 has peptide ions at two mass units lower (−2 u) than the untreated protein which, based on the MS2 spectra (Fig. 6 and S3A and B†), are assigned to peptide T6 and T8 that have lost an H atom (−1 u) from each of two tyrosines. Notably, no bn or yn sequence ions arising from peptide-bond fragmentation between the oxidized tyrosines appear in the MS2 spectra (Fig. 6B and S3B†). In fact, the stability of the cyclic peptide region identifies Y36–Y39 and Y36–Y42 as crosslinks in T6 (Table S3†). The yield of crosslinked T6 and T8 is >70% in overoxidized Ccp1 (Fig. 5B) and, in addition to M230/M231 oxidation (Fig. 3A and Table S3†), ∼10% of Y229–Y236 undergoes crosslinking in T26 (Fig. 5B and S3D†) in competition with Y229 hydroxylation (Fig. 5A). Intramolecular dityrosine crosslinking has not been reported for overoxidized Ccp1 previously but intermolecular crosslinking involving the T6 tyrosines (Y36, Y39, Y42)22,34,35 and Y23623 is documented. Presumably, the Ccp1 dimers and trimers detected here (Fig. S2A†) contain such intermolecular crosslinks.
Fig. 6 LC-MS/MS analysis of dityrosine formation in tryptic peptide T6. MS2 spectrum of the (M + 3H)3+ ion of: (A) native T6 at m/z 672.9784 and (B) oxidized T6 at m/z 672.3047. The T6 precursor ions (green) were fragmented by CID (30 V) to give bn (red) and yn (blue) sequence ions. The y102+ and y132+ ions encircled in panel B have masses consistent with loss of an H atom (−1 u) from both Y36 and Y39. The peptide sequence in each panel shows Y36, Y39 and Y42 in red font and the observed fragmentations are mapped onto the sequence. Note the absence of fragmentation between crosslinked Y36 and Y39 in panel B. For clarity, low abundance ions are not mass labeled in the spectra but a complete list of the identified sequence ions is provided in Tables S6 and S7.† |
Fig. 7 The proximal heme ligand H175 and the distal H52 are oxidized to HisO. (A) Yield of HisO formation. Experimental details are given in the caption to Fig. 3, and Fig. S5† shows the MS2 spectra of T7 and T21 with oxidized H52 and H175. (B) UV-vis spectrum of 1 μM Ccp1 treated with 0 (black trace), 1 (blue trace) and 10 M eq. of H2O2 (green trace). Spectra were recorded at pH 8.1 in KPi/DTPA 1 h after H2O2 addition. Results in panel B are representative of three independent experiments. |
Close to 60% of the distal H52 is present as HisO in Ccp1 oxidized with 10 M eq. of H2O2 (Fig. 7A). The low CCP activity (11%) of this sample (Table S4†) reflects the critical function of H52 as an acid–base catalyst in heterolytic cleavage of the peroxy bond of H2O2 as evinced by the 105-fold lower H2O2 reactivity of the H52L variant.48 The extensive heme loss in Ccp1 exposed to 100 M eq. of H2O2 (Fig. S4E†) may result from attack by the OH˙ produced on homolytic H2O2 cleavage catalysed by the heme or heme-derived iron following H52 oxidation.
Free methionine and many methionine residues are oxidized to MetO with H2O2 as a typical oxidant.65 Nonetheless, MS analysis provides no convincing evidence for more MetO formation in H2O2-treated apoCcp1 than in the untreated apoprotein (Fig. 2Dvs.A). This we attribute in part to inhibition of trace-metal activation of H2O2 by the 100 μM DTPA present in the buffer. Tryptic digestion was also performed in the presence of DTPA but 2–20% MetO is detected in peptides from untreated holoCcp1 (Fig. 3A), which may signal methionine oxidation catalyzed by the heme released during proteolysis. With the possible exception of M163, MetO levels increase significantly in peptides from oxidized Ccp1 (Fig. 3). Hole hopping from the oxidized heme to methionine residues in the intact protein followed by reaction of the resultant radical with O2 could generate MetO on water capture (Scheme S5†).63,66 Such heme-mediated methionine oxidation has been reported previously in the autoreduction of the H2O2-oxidized di-heme of MauG. This is coupled to the oxidation of a nearby methionine to MetO and an intermediate methionine radical is assumed to be stabilized by a two-center, three-electron (2c3e) bond between the sulfur atom and an amide nitrogen or oxygen or an aromatic group.67
Hole transfer to cysteine should be more thermodynamically favorable than to methionine (Table 1). Also, free C˙ reacts rapidly with O2 to give a peroxyl radical (CysSOO˙) that has been detected by EPR (Table 1). Superoxide release and water capture would give CysOH (Scheme S4†) but C128 conversion to CysO2H and CysO3H (Fig. 3B) may not be all heme-mediated given the known instability of sulfenic acids to further oxidation.
Neutral W˙ also is readily converted to a peroxyl radical by O2 (Table 1). Again, superoxide release and water capture by the aryl carbocation would lead to TrpOH, with indole-ring hydroxylation at the 2-, 4-, 5-, 6-, or 7-positions (Scheme S1†). TrpOH can be further oxidized to Trp(OH)2,68 and ∼30% of W223 is detected as kynurenine (a tryptophan metabolite)69 in overoxidized Ccp1 (Fig. 4B, inset). Although the pKa of free W˙+ is ∼4 (Table 1), W191˙+ is stabilized in CpdI,70 allowing hole hopping from this site to nearby solvent-exposed residues at the protein surface, including W211 and W223 (Fig. 8). Radicals on these tryptophans are scavenged by O2 (Scheme S1†) as evidenced by their oxidation to TrpOH and Trp(OH)2 (Fig. 4). Distal W57 is also solvent exposed whereas a number of internal waters are <5 Å from W51 and W126 (Fig. 8 and Table S8†) to accept a proton from their W˙+ form and promote their 100% conversion to Trp(OH)2 and TrpOH, respectively, in overoxidized Ccp1 (Fig. 4).
Fig. 8 Zoning of Ccp1 based on mapping of the oxidized residues onto its structure. PyMOL-generated cartoon of Ccp1 structure (PDB 1ZBY) with labels on the 24 residues (W, blue; Y, green; H, orange; M, grey; C, magenta) oxidatively modified on reduction of H2O2 at the heme. The oxidized residues are assumed to be the termination sites of hole hopping from the heme. Hole transit through W191 oxidizes residues in zone 1 (blue) until M230/M231 oxidation turns on additional pathways from the heme to zones 2a and 2b (pink). As these pathways become exhausted, residues close to the heme (zone 3; green) are oxidized. Not much hole termination is detected in zone 4 (yellow) since only one (Fig. 5A) of its four tyrosines (, Y203, , ) is oxidized. See text for further discussion of the proposed hole-hopping pathways. Solvent-exposed residues are underlined and 31 conserved internal water molecules (see Fig. S6†) are depicted as pink spheres. |
In contrast to W˙, the slow reactivity of Y˙ with O2 is noteworthy (Table 1 and Scheme S2†). However, Y˙ radicals rapidly dimerize and dityrosine is the dominant tyrosine oxidation product in overoxidized Ccp1 (Fig. 5). The side chains of Y36, Y39 and Y42 are separated by 4–8 Å on the same α-helix and Y36/Y39 and Y39/Y42 crosslinks are found in T6 (Table S3†). Y67 and Y71 are 10 Å apart in a large loop region (Fig. 8) with sufficient flexibility for dityrosine formation as evidenced by the effective crosslinking in T8 (Fig. 5B and S3B†). Remarkably, extensive crosslinking of its distal region causes negligible change in Ccp1's secondary structure as assessed by circular dichroism (Fig. S7†).
The efficient (∼80%) hydroxylation of Y229, which may be a consequence of its proximity to M230, results in the only TyrOH formed in high yield (Fig. 5A). In fact, half of Ccp1's tyrosines (Y16, Y23, Y153, Y187, Y203, Y244, Y251) appear to undergo little or no oxidation. The thermodynamics of Y˙ formation require deprotonation because of the low pKa of Y˙+ (Table 1), but all 14 tyrosines are solvent exposed (Fig. 1) and/or close to internal waters (Fig. S6 and Table S8†) so lack of a proton acceptor is unlikely a deciding factor in Y˙+ formation. Advantageously, the presence of several unmodified tyrosines in overoxidized Ccp1 aids in mapping hole-hopping pathways from the heme and in delimiting donor zones within the polypeptide (Fig. 8).
Since the pKa of free H˙+ is ∼5–7 and H˙+ has an E7 of 1.17 V (Table 1), oxidation of histidine residues also will be sensitive to the local protein environment. Hydrogen bonding to D235 imbues H175 with imidazolate character, which promotes donation of electron density to the hypervalent heme.73 However, we detect <5% H175 oxidation in Ccp1 treated with 1 M eq. of H2O2 (Fig. 7A) but this increases to ∼50% in overoxidized Ccp1 as discussed in the next section. The stable HisO product is likely formed via a peroxyl radical (Scheme S3†) as proposed for the other oxidizable residues.
Oxidation of M230 and/or M231 converts W191 into a poorer electron donor as seen on mutation of these residues.17,18 Hence, hole hopping from the heme to the distal region (zones 2a and 2b, Fig. 8) opens up. Both W126 and C128 act as major distal donors in overoxidized Ccp1 (Table S5†). These neighboring residues are in a favorable environment for hole formation and subsequent termination via O2 scavenging (Schemes S1 and S4†), being close to 2–3 internal waters and O2 docking sites (Fig. S6 and Table S8†). W57 and Y67/Y71 are additional termination sites in zone 2a whereas zone 2b contains solvent-exposed Y36, Y39 and Y42 that undergo efficient hole termination by crosslinking once oxidized to Y˙ (Fig. 5B and Scheme S2†). The sequence of hole hopping to zones 2a and 2b is not resolved in our study but the clustering of donors into these subzones suggests two distinct routes from the heme via transient hole formation on W51 and on M119, respectively (Fig. 8).
W51, W191 (Fig. 4) and H52 (Fig. 7) kick in as major endogenous donors in Ccp1 treated with 10 M eq. of H2O2. Hole-hopping from the heme presumably terminates at these active-site residues following oxidation of residues further from the heme. We group W51, W191, H52 and M119 into zone 3 (Fig. 8) together with M172 and H175, which are maximally oxidized (∼50%) in Ccp1 exposed to 5 M eq. of H2O2 (Fig. 3A and 7A). Significantly, M172 and H175 are, for the most part, oxidized in different Ccp1 molecules (Table S5†). Also, the relative orientation of H175 and W191 is fixed by hydrogen bonds to D235, which additionally modulate coupling of W191˙+ to the heme.73 Thus, the susceptibility to oxidation of these three proximal residues must be interdependent. Given its probable critical importance in labilizing the heme,14 computational investigations of H175 oxidation, including the roles of M230 and M231 that stabilize W191˙+,9,17,46 are underway. Oxidation of the distal H52 seen in the 10:1 H2O2:Ccp1 sample (Fig. 7A) also is a vital step since this histidine is essential for H2O2 heterolytic cleavage by Ccp1.48 Shutting-down this rapid reaction by H52 oxidation is necessary to defend the heme as the endogenous donors become exhausted.
So far we have only considered hole hopping from the heme as a mechanism of residue oxidation in Ccp1. For the sake of completeness, we note that once H52 is oxidized (Fig. 7), slow homolytic H2O2 cleavage may generate some ˙OH at the heme. A likely target would be W51 just above the heme (Fig. 8) as highly reactive ˙OH radicals do not diffuse far from their site of generation. It is noteworthy that W51 is 100% dihyroxylated in the 10:1 H2O2:Ccp1 sample (Fig. 4B), which may reflect some oxidation of this residue by ˙OH. However, we anticipate very little ˙OH formation in the H2O2–Ccp1 incubations examined here since H2O2 is consumed rapidly.32 In CuZn-superoxide dismutase, on the other hand, homolytic cleavage of H2O2 at the catalytic copper results in oxidation of histidines ligated to the catalytic center.62,74,75 In fact, histidines are oxidized in the dismutase during cell aging,76,77 and HisO is viewed as a biological marker for evaluating protein modification from oxidative stress.78
The assignment of W˙ radicals by EPR can be challenging due to peak broadening.81 Moreover, their efficient scavenging by O2 as seen in oxidized Ccp1 (Fig. 4) will compete with their detection by EPR. In fact, W191˙+ with low O2 reactivity is the only indolyl radical reported in EPR studies of CpdI and overoxidized Ccp1.16–21 Furthermore, MNP succeeded in trapping Y153˙, Y39˙ and Y236˙ but no W˙ radicals in Ccp1,22,23 revealing that spin trapping also is biased toward Y˙ with low O2 reactivity. Thus, the current results underscore the complementarity of MS and EPR for studies of protein-based radicals. EPR can directly detect relatively long-lived radicals and provide information on their stability and, in some instances, the specific residues oxidized can be selected from EPR spectra.81 MS, on the other hand, can identify all oxidized residues and additionally characterize their stable end products. This sheds light on radical-quenching mechanisms and also on possible hole-hopping pathways from heme or other redox-active metal centers in proteins.
In addition to confirming W191˙+ as the main radical species in CpdI, a recent QM/MM computational analysis82 proposed Y203/Y251 and Y236 as secondary radical sites in two possible pathways from W191˙+. Y203/Y251 together with Y153 and Y244 are clustered in proximal zone 4 (Fig. 8) and undergo little or no oxidation detectable by MS (Fig. 5). However, we did detect TEMPO mass adducts of peptides that contain tyrosines from zone 4 (T18, T23, T27 + 28, and T28),26 and a MNP mass adduct has been localized on Y153.22 Thus, the mass adducts found by MS or the spin adducts detected by EPR will depend on the trapping and scavenging agents employed as well as on the agents' accessibility to different protein regions. In this context, it is pertinent to point out that scavenging of protein-based radicals by O2 is of physiological relevance due to its presence in cells under aerobic conditions.
Intriguingly, ascorbate peroxidase (APX) possesses a very similar active-site structure to Ccp1 with W41 and W179 occupying the same distal and proximal locations as W51 and W191, respectively, in Ccp1.87,88 However, heme crosslinking to W41 occurs in oxidized APX,88 which is associated with stabilization of a transient π-cation radical on the porphyrin rather than on W179.87 Ccp1W191F also forms a transient porphyrin π-cation radical and heme crosslinking to W51 has been reported in this variant.89 Thus, ultra-rapid radical transfer from the porphyrin appears to be key in preventing heme crosslinking to the distal tryptophan in Ccp1. A role other than that of catalytic H2O2 scavenger has not been proposed for APX whereas our recently published targeted proteomics study provides additional support for Ccp1's participation in heme transfer.90
Intramolecular dityrosine crosslinking is prevalent in Ccp1 overoxidized with 5 and 10 M eq. of H2O2 (Fig. 5B). Crosslinked Ccp1 may signal oxidative stress in yeast given that dityrosine is becoming increasingly identified as a marker of oxidative stress and is linked to a number of pathologies including amyloid fibril formation.91
Interestingly, Fe-catalyzed histidine oxidation is implicated in H2O2 sensing by the peroxide resistance protein (PerR) from B. subtilis. H2O2 binding to the non-heme FeII center of PerR results in oxidation of the Fe ligands (H37 and H91), promotes Fe release and apoPerR dissociation from DNA to turn on genes such as that encoding the catalase, KatA.93,94 Thus, both PerR and Ccp1 can be viewed as metalloreceptor proteins that enable their ligand, H2O2, to regulate its own consumption by a catalase: H2O2 regulates heme transfer from Ccp1 to apoCta1 and Fe loss from PerR to initiate KatA translation. Although these mechanisms are fascinatingly different, they share a common essential step: oxidation by H2O2 of Fe-ligated histidine to trigger heme or Fe release from the metalloreceptor.
Finally, on a methodological note, we reiterate that our detailed analysis of oxidized Ccp1 using a high-performance universal detection/characterization method such as MS exposes a bias in EPR detection toward protein-based radicals like Y˙ with low O2 reactivity. This should be borne in mind when mapping hole-hopping pathways in proteins based on EPR monitoring of transient radicals.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc03125k |
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