# Methionine oxidation of Carbohydrate-Active enZymes during white-rot wood decay

Lise Molinelli, Elodie Drula, Jean-Charles Gaillard, David Navarro, Jean Armengaud, Jean-Guy Berrin, Thierry Tron, Lionel Tarrago

<figure><img src="image_1.png" /></figure>

## To cite this version:

Lise Molinelli, Elodie Drula, Jean-Charles Gaillard, David Navarro, Jean Armengaud, et al., Methionine oxidation of Carbohydrate-Active enZymes during white-rot wood decay. 2023. hal-04266319

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Distributed under a Creative Commons Attribution 4.0 International License
19 Abstract

20 White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen
21 species (ROS), like hydrogen peroxide ($H_2O_2$), to degrade lignocellulose in wood. $H_2O_2$ serves as a co-
22 substrate for key oxidoreductases during the initial decay phase. While the degradation of
23 lignocellulose by CAZymes is well-documented, the impact of ROS on the oxidation of the secreted
24 proteins remains unclear and the identity of the oxidized proteins is largely unknown. Methionine
25 (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO₂) with potential deleterious,
26 antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using
27 the white-rot model *Pycnoporus cinnabarinus* grown on aspen wood, we analyzed the Met content of
28 the secreted proteins and their susceptibility to oxidation combining labelled $H_2^{18}O_2$ with proteomics.
29 Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins
30 (2.1%), a feature conserved in fungi but not in other eukaryotes. We also evidenced that a catalase,
31 widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics
32 approach allowed identification of 49 oxidizable Met and 40 oxidizable Pro residues within few
33 secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized
34 in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a
35 substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting
36 important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins
37 by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in
38 CAZymes.
39

39
40

**Importance**

41 The study of lignocellulose degradation by fungi is critical for understanding the ecological and
42 industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-
43 established role in lignocellulose degradation, the impact of hydrogen peroxide ($H_2O_2$) on secreted
44 proteins remains unclear. This study aims at evaluating the effect of $H_2O_2$ on secreted proteins,
45 focusing on the oxidation of methionine (Met). Using the model white-rot model *Pycnoporus*
46 *cinnabarinus* grown on aspen wood, we showed that fungi protect their secreted proteins from
47 oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous $H_2O_2$.

48 The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met like those
49 of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in
50 lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot
51 fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these
52 processes in lignocellulose breakdown.

53
54 **Introduction**

Filamentous fungi are crucial in the carbon cycle because of their ability to decompose organic matter and release carbon back into the ecosystem (1). Wood-decay fungi extract carbon from recalcitrant polymers like cellulose and hemicelluloses embedded in a lignin matrix, by secreting enzymes into the extracellular environment (2). The secreted proteins, commonly referred to as the "secretome", contains the carbohydrate-active enzymes (CAZymes), including large sets of glycoside hydrolases (GH) and auxiliary activity (AA) enzymes (3, 4). The GH class contains the endo- and exo-acting enzymes targeting cellulose and hemicelluloses. The AA class contains laccases (AA1) and class II peroxidases (AA2) involved in lignin degradation, and lytic polysaccharide monooxygenases (LPMO, AA9) proposed to play an important role in the early phase of wood degradation (5-7). Reactive oxygen species (ROS), particularly hydrogen peroxide ($H_2O_2$), play a crucial role in the early phase of wood decay (8-10). $H_2O_2$ is generated by glucose-methanol-choline oxidoreductases (AA3) and copper radical oxidases (AA5), that target carbohydrate or aliphatic/aromatic alcohols (11, 12). In white-rot fungi, $H_2O_2$ acts as a co-substrate to fuels different types of LPMOs and AA2 peroxidases (13-15). While the roles of CAZymes and $H_2O_2$ during the degradation of lignocellulose is well-documented, the impact of ROS on the oxidation of CAZymes remains unclear. Fenton-generated ROS were shown to decrease enzymatic activity of some GHs in white-rot secretomes, through the oxidation of the secreted proteins, and the most abundant form of oxidation found was the oxidation of Met residues (16). Met can be converted by $H_2O_2$ into Met sulfoxide (MetO) or Met sulfone (Met$O_2$) and this has been shown to play critical roles in many organisms (17-20). For instance, Met oxidation can lead either to a loss of function or act as a regulatory switch, to activate enzymes (21, 22). The presence of oxidizable Met in proteins can also be associated to an antioxidant role by serving as a protection to avoid the oxidation of residues critical for the protein function (23). MetO can be reduced back to Met by the methionine sulfoxide reductases, but, as these enzymes are most likely not secreted they cannot reduce oxidized Met of secreted proteins (24). Regarding the importance of $H_2O_2$ during wood decay, our hypothesis is that some Met of secreted proteins can be oxidized and play antioxidant or regulatory
80 roles. Interestingly, the treatment of an α-galactosidase (GH27) from *Trichoderma reesei* with H₂O₂
81 strongly increased its activity due to the oxidation of a Met located near the active site, suggesting that
82 Met oxidation of CAZymes can have a positive effect on their activity (25). To identify secreted proteins
83 with oxidizable Met, we selected the white-rot *Pycnoporus cinnabarinus* grown on aspen wood as a
84 model. *P. cinnabarinus* secretes H₂O₂-producing enzymes in the early phase of wood degradation, to
85 provide co-substrate for the H₂O₂-consuming CAZymes (26), but also as potential oxidant of Met. We
86 characterized the secreted proteins and investigated the sensitivity of their Met to oxidation by using
87 a redox proteomics approach based on the use of ¹⁸O-labeled hydrogen peroxide (H₂¹⁸O₂) (27–30). Our
88 investigations reveal the extend of Met oxidation in fungal secretomes and the precise localization of
89 oxidizable residues in secreted proteins and CAZymes, suggesting potential antioxidant or regulatory
90 roles.

91
rate of 0.25 µL per min. Data-dependent acquisition analysis of the peptides was performed with an Orbitrap Exploris 480 tandem mass spectrometer (Thermo) as described (32). Each full scan of peptide ions in the high-field Orbitrap analyzer was acquired from m/z 350 to 1800 at a resolution of 120,000, selecting only 2+ and 3+ charged precursors for high-energy collisional dissociation and with a dynamic exclusion of 10 seconds. MS/MS spectra of fragmented precursors were acquired at a resolution of 15,000 and assigned to peptide sequences by the MASCOT Server 2.5.1 search engine (Matrix Science) using the *P. cinnabarinus* annotated proteome, with fixed and variable modifications (Datasets 3; 4). Proteins were validated when at least two different peptides (p-value <0.01) were detected, resulting in a protein identification false discovery rate below 1%. Protein abundance was estimated using the Normalized Spectral Abundance Factor (NSAF) (33). The Met content of the proteins was calculated using the entire sequence. The Met content corrected by the NSAF was calculated using the following formula:

$$ \frac{\sum(\%Met_{protein n} \times NSAF_{protein n})}{\sum NSAF_{protein n}} $$

With %Met<sub>protein n</sub> and NSAF<sub>protein n</sub> corresponding to the percentage of Met and the NSAF value of the individual proteins identified, respectively. The percentage of modification of Met or Pro residues was calculated by dividing the number of spectral counts (SC) of the residue with the modification by the total number of SC for the residue and then multiplying the result by 100. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (34) partner repository with the dataset identifier PXD046271 and 10.6019/PXD046271.

### H₂O₂-consuming activity

Secreted proteins (20 µg) were incubated 1 h with H₂O₂ (30 mM) in sodium acetate buffer, pH 5.2. A control without proteins was done. Then, the samples were filtered using Vivasin 3 kDa cut-off
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and the remaining H₂O₂ in filtrates was titrated by absorbance measurement at 240 nm and compared to H₂O₂ concentration in controls.

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142

143

**Analysis of Met content in P. cinnabarinus, fungi, metazoans, and plants proteins.**

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P. cinnabarinus proteins were predicted secreted if they fulfilled three conditions: (i) presence of a secretion signal peptide, (ii) absence of endoplasmic reticulum retention motif and (iii) absence of transmembrane helix outside the signal peptide, as described (26). Experimentally validated fungal secreted proteins were retrieved from proteomics analyses (7, 35–49), from Uniprot (www.uniprot.org) and FunSecKB2 (50). Metazoans experimentally validated secreted were retrieved from Uniprot, MetazSecKB (51) and the Human Protein Atlas (52, 53). Experimentally validated plant secreted proteins were retrieved from Uniprot, PlantSecKB (54) and the SUBA database (55). The datasets of fungal, metazoans and plants non-secreted proteins were made using proteins retrieved from Uniprot, the Human Protein Atlas and the SUBA database. For all datasets, only manually curated proteins, and those found with at least 5 SC in proteomics, were conserved. Membrane proteins, proteins shorter than 50 amino acids, or not starting with Met were discarded. Statistical analyses were unpaired t-test with Welch's correction.

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**Conservation of proteins and oxidized residues.**

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A hundred and twenty fungal genomes were selected based on their public availability and their representativity in the 9 fungal phyla (56, 57). Targets of the oxidized proteins were searched using BLASTP (v 2.6.0+) (58). The 100 sequences with the highest e-values were aligned using MAFFT tool (version 6.864b) (59). The conservation of individual residues was made by counting its occurrence in all the sequences.

163
164 **Results**

165 **Optimized methods allowed to obtain secreted proteins representative of early days decay.**

166 To identify oxidizable Met in the secreted proteins of *P. cinnabarinus*, we focused on the early
167 days of wood decay, during which production and consumption of H₂O₂ are likely to occur (42). We
168 performed a time-course growth of the fungus on aspen wood and purified the proteins from the
169 culture supernatants at days 3, 5 and 7 on successive anion and cation exchange chromatography to
170 avoid artifactual oxidation. Then, we oxidized the secreted proteins with H₂¹⁸O₂ and identified them
171 and their potential oxidative Met modifications using proteomics (Fig. S1). We identified 308 secreted
172 proteins, among which CAZymes and proteases represented 41% and 10%, respectively (Fig. 1; Table
173 S1; Dataset 1). Among the CAZymes, GH was by far the most represented class, with 89 proteins (71%)
174 spread over 34 families (Fig. 1; Table S1). We identified most of the CAZymes expected during wood
degradation, i.e. cellulases (GH3, GH5_5, GH5_9 and GH7, GH12, GH45), hemicellulases (GH10, GH43,
175 GH51, CE15, CE16) (4), as well as pectinases (GH28, CE8) and also some CAZymes targeting fungal cell
176 wall components (e.g. GH18 chitinases) (Table S1). Out of 23 identified AAs (18%), we found laccases
177 (AA1) and class II peroxidases (AA2), LPMOs (AA9) and H₂O₂-generating enzymes (AA3, AA5). We
178 estimated the abundance of the individual proteins using the Normalized Spectral Abundance Factor
179 (NSAF) (Dataset 1). The mean %NSAF of all identified proteins was ~ 0.3% and 52 proteins were above
180 this value. The most abundant protein categories were CAZymes (52%), proteins of unknown function
181 (35%) and proteases (8%). Among the most abundant CAZymes, we found the three cellobiohydrolases
182 (GH7), three chitinases (GH18) and two laccases (AA1). Interestingly, three H₂O₂-generating enzymes
183 (two AA3_2 and one AA5_1) were among these most abundant CAZymes (Dataset 1). Overall, the
184 identity of the secreted proteins and their abundance validated our approach, indicating that the
185 growth conditions, the proteins preparation method, and the oxidative treatment used were suitable
186 for studying Met sensitivity to oxidation.

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<caption>**Figure 1.** Distribution of the identified secreted proteins and CAZymes. Values shown are the aggregate of all replicates for each day (Table S1; Dataset 1). Bar graphs represent the number of CAZymes per class.</caption>

195
196 *Met content is strongly reduced in fungal secreted proteins.*

197 First, we evaluated the Met content of the secreted proteins, which was ~1.4% for each time-
198 point (Fig. 2A). Then, to determine whether some abundant proteins with specific Met content could
199 influence the overall content, we corrected the values by the proteins NSAF. The corrected Met
200 contents were 1.21 ± 0.02, 1.33 ± 0.02, and 1.35 ± 0.02, for days 3, 5, and 7, respectively (Fig. 2A).
201 These results indicate that the Met content in the proteins secreted by *P. cinnabarinus* is comprised
202 between 1.2 to 1.4%. Strikingly, this value is twice lower than the Met content of proteins in general,
203 i.e. 2.4% (60). We investigated whether this characteristic may apply to all secreted proteins of *P.
204 cinnabarinus*. The theoretical *P. cinnabarinus* secretome consists of 666 proteins with an average Met
205 content of 1.55 ± 0.82%, while the fungus non-secreted proteins have a higher mean of 2.07 ± 0.96%
206 Met. (Fig. 2B; Table S2; Dataset 2). These results show that *P. cinnabarinus* secreted proteins contain
207 significantly less Met than the non-secreted. Then, we compared the Met content of experimentally
208 validated secreted proteins from fungi, metazoans, and plants with the Met content of intracellular
209 proteins (Fig. 2B; Table S2; Dataset 2). Fungal secreted proteins have a mean Met content of 1.52 ±
210 0.74%, significantly lower than that of non-secreted proteins (2.17 ± 0.99%). In contrast, the Met
211 content of metazoans secreted proteins (2.68 ± 1.40%) is significantly higher than that of the non-
212 secreted proteins (2.41 ± 1.07%). No statistical difference exists between the Met content in secreted
213 (2.59 ± 1.55%) and non-secreted (2.55 ± 1.14%) proteins of plants (Fig. 2B; Table S2; Dataset 2).
214 Altogether, these results show that the secreted proteins of fungi contain less Met than the
215 intracellular proteins, and that this feature is not conserved in other eukaryotes.

216
<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /></figure>

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**Figure 2.** Met content of *P. cinnabarinus* secreted proteins and comparison with fungal, metazoan, and plant secreted and intracellular proteins. A) Met content of the identified *P. cinnabarinus* secreted proteins calculated for each protein and averaged for the three secretomes at each time point. Alternatively, the Met content for each protein was normalized by the protein NASF and averaged. Values shown are the mean of the 3 replicates ± SD. B) Met content of intracellular proteins and of secreted in the *P. cinnabarinus* theoretical proteome versus the Met content of experimentally validated intracellular and secreted proteins from fungi, metazoans, and plants (Table S2; Dataset 2). Values shown are the mean of all proteins in each set ± SD. Statistical analysis performed by unpaired t-test with Welch's correction (*P* ≤ 0.001; n.s., not significant).

227
229 The identification of oxidizable Met in P. cinnabarinus secreted proteins required accurate
230 quantification of their oxidation levels. H₂¹⁸O₂ was used to prevent artifactual Met oxidation potentially
231 occurring during sample preparation by blocking the Met reduced in the samples and converting them
232 to Met sulfoxide or sulfone containing ¹⁸O atom (Met¹⁸O and Met¹⁸O₂, respectively). The use of a
233 concentrated H₂¹⁸O₂ solution (100 mM) should allow the oxidation of all the reduced Met and the
234 reliable quantification of the initial levels of Met¹⁶O and Met¹⁶O₂ (28, 29). For the day 3 secretome, the
235 levels of Met carrying ¹⁸O or ¹⁶O, either as sulfoxide or sulfone, were ~64% and ~18%, respectively.
236 Although, ~18% of Met in reduced form were still present, this indicated that most Met from the
237 identified proteins were oxidized after the treatment with H₂¹⁸O₂. Of note, ¹⁶O or ¹⁸O sulfone levels
238 reached ~2% (Fig. 3A; Table S3; Dataset 3). On the contrary, in day 5 and day 7 secretomes, the levels
239 of MetO and MetO₂ carrying ¹⁸O were extremely low (~1%), indicating that the blocking strategy failed
240 for these samples (Fig. 3A; Table S3; Dataset 3). To evaluate whether this lack of oxidation by H₂¹⁸O₂
241 in secretomes of days 5 and 7 affected only Met residues, we reanalyzed our data by searching for two
242 main forms of Pro carbonylation bearing ¹⁸O. For the day 3 secretome, we found slightly less than 4%
243 of Pro oxidized with ¹⁸O. A similar content of ¹⁶O was found, indicating that the large majority of Pro
244 were not oxidized (~92%) (Fig. 2B; Table S4; Dataset 4). This low level of Pro oxidation was anticipated
245 due to its low reactivity with H₂¹⁸O₂ (61). The percentage of ¹⁸O oxidation decreased to ~0.5% in the
246 day 5 and day 7 secretomes, while ¹⁶O oxidation levels remained comparable to those observed for
247 the day 3 secretome. Thus, the trend for Pro is the same as for Met indicating that the lack of oxidation
248 observed for Met was not specific to this residue. Proteins secreted during the days 5 and 7 of decay
249 were not oxidized by exogenous H₂¹⁸O₂, unlike those secreted at day 3. These results prompted us to
250 search for secreted enzymes that scavenge H₂O₂. A catalase stood out: almost absent at day 3 (0.02 ±
251 0.02 %NSAF), but more abundant at days 5 and 7, with %NSAF values of 0.33 ± 0.05 and 0.42 ± 0.01,
252 respectively (Fig. 2C; Dataset 1). When we measured the H₂O₂-consuming activity of the secretomes,
253 we observed no activity at day 3, but strong activities at days 5 and 7 secretomes, in a perfect
consistency with the catalase abundance (Fig. 2C). Although few potential peroxidases and LPMOs were present, there was no difference in their abundance during the time course (Dataset 1). These results indicated that this catalase was very likely responsible for most, if not all, of the H₂O₂-consuming activity. Altogether, these results show that, under our experimental conditions, *P. cinnabarinus* secreted a catalase at days 5 and 7, which, by consuming potential exogenous H₂O₂, protected the secreted proteins from oxidation. On the contrary, at day 3, the secreted proteins are left without any protection against exogenous oxidant. On the other hand, this highlight that the strategy consisting of using high concentrations of H₂¹⁸O₂ to block reduced Met can hardly be applied to the secretomes collected at days 5 and 7, at least in conditions where hydroxylamine was not an efficient inhibitor of H₂O₂-consuming activity. Nonetheless, since Met oxidized with ¹⁸O were detected, we analyzed the nature of the oxidized proteins to identify Met residues with potential antioxidant or regulatory roles.

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<figure><img src="image_6.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_8.png" /></figure>

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**Figure 3.** Levels of Met and Pro oxidation in the *P. cinnabarinus* secretomes and H₂O₂-consuming activity. A) Total levels of Met oxidation as sulfoxide and sulfone, either ¹⁶O or ¹⁸O calculated using the spectral counts of all Met-containing peptides and compared to the level of non-oxidized Met for each time point (**Table S3; Dataset 3**). B) Total levels of Pro oxidation as glutamic 5-semialdehyde (G5S) and pyroglutamic acid (PA), either ¹⁶O or ¹⁸O calculated using the spectral counts of all Pro-containing peptides and compared to the level of non-oxidized Pro for each day (**Table S4; Dataset 4**). C) %NSAF of the catalase A0A060STN2 (**Dataset 2**) and total H₂O₂-consuming activity at each time point.

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275 *Most of the oxidations occur on a few secreted CAZymes.*

276 Among the 308 secreted proteins identified, we detected 265 Met belonging to 118 proteins
277 (Table S3; Dataset 3). We selected only the Met-containing peptides present in all replicates for each
278 secretome, with at least five spectral counts, in at least two out of the three replicates. At day 3, all
279 selected Met were found oxidized with ¹⁸O (Fig. 4A; Dataset 3). The total oxidation (¹⁶O and ¹⁸O) ranged
280 from ~20% to 100% for the 46 identified Met (Fig. 4A). Four Met, all from CAZymes, were fully oxidized
281 and thus saturated with ¹⁸O: Met305 of a PL35, Met378 of a GH7, Met314 of a PL8, and Met239 of a
282 CE16 (Fig. 4A; Dataset 3). Twenty-four proteins had oxidation levels ranging from ~81% to 97% and
283 17 from 57% to ~80%. Only the Met592 from an AA3_2 displayed a much lower value of total oxidation
284 (~20%) (Fig. 4A; Dataset 3). The quantification of the ¹⁶O-oxidation levels appeared clear for the four
285 ¹⁸O-saturated Met and should correspond to the levels "naturally" found during the wood decay.
286 However, for all other Met with an oxidation value less than 100%, the saturation with ¹⁸O was
287 incomplete and some artifactual oxidation may have occurred during the sample preparation, and thus
288 the level of ¹⁶O might be overestimated. For these proteins, we assumed that the percentage of ¹⁶O
289 found during wood decay was necessarily equal to or lower than the measured one. The percentage
290 of oxidation as ¹⁶O ranged from 0% to 47% with a mean of 18% (Fig. 4A; Dataset 3). This value is
291 consistent with the mean percentage of Met¹⁶O oxidation calculated considering all Met containing-
292 peptides found (see Fig. 3A) and indicated that most of the oxidation was concentrated on these few
293 secreted proteins. Concerning day 5 and day 7 secretomes, the very low levels of ¹⁸O oxidation
294 precluded the use of the blocking strategy. However, to find proteins with Met¹⁸O may allow to
295 consider H₂¹⁸O₂ as a probe for highly reactive residues, because of the low H₂¹⁸O₂ concentration they
296 were exposed to, due to the catalase activity. We found eight and nine Met bearing ¹⁸O in the proteins
297 secreted at day 5 and day 7, respectively, and five were common to both samples (Fig. 4B; Dataset 3).
298 The percentages of ¹⁸O ranged from ~2% to ~14%, and from ~1% to ~23%, for the secreted proteins
299 of day 5 and day 7, respectively. Among the 11 oxidized Met, only three were uniquely found at day 5
300 or day 7: Met533 of a GH95 at day 5, and Met284 and Met160 belonging to a PL35 and a GH3,
respectively, at day 7 (Fig. 4B; Dataset 3). This indicates that most of the proteins found oxidized with ¹⁸O at day 5 or 7 were also oxidized at day 3, highlighting their high reactivity to H₂O₂. We found a total number of 49 oxidizable Met belonging to 28 proteins, representing ~24% and ~19% of all the identified Met and proteins from the secretome, respectively (Fig. 4C; Dataset 3). Interestingly, the 18 identified CAZymes represented most of them (64%), while proteases and proteins of unknown function both represented 18% with five identified proteins. The most represented CAZymes were GHs with 10 identified proteins. These results indicate that the proportion of oxidized CAZymes was enriched compared to the different types of proteins present in the secretomes (compare Fig. 4C with Fig. 1).

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<figure><img src="image_9.png" /><figcaption>Figure 4. Identity of oxidized Met and their levels of oxidation in *P. cinnabarinus* secretomes. A)</figcaption></figure>

Proportion of the different forms of oxidation for the Met identified in the secreted proteins at day 3.

Proteins are ranked based on the percentage of ¹⁶O (MetO and MetO₂). The four Met fully oxidized are

underlined. Values shown are the mean of oxidation without standard deviation, omitted for clarity.

All values are in **Dataset 3. B) Proportion of the different forms of oxidation for the Met identified in**

the secreted proteins at day 5 and day 7. Proteins are ranked based on the percentage of ¹⁸O (MetO

and MetO₂). All values are in **Dataset 3. C) Distribution of the identified secreted proteins and CAZymes**

with oxidized Met. Values shown are the aggregate of all replicates for each time point (**Dataset 3**).

Bar graphs represent the number of CAZymes per class.
323

Oxidation occurs in multiple sites and hotspots of homologous proteins.

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Interestingly, among the five most oxidized, three Met belong to the three GH7 (A0A060SFM6, A0A060SMU8, A0A060ST00, named A, B, and C, respectively), at position 286 of the GH7 A and B, and at position 287 of the GH7 C (Fig. 4A). A sequence alignment showed that these positions are conserved between these GH7 homologs (Fig. 5A). These Met were oxidized with similar levels of Met¹⁶O (between 28% and 38%) and Met¹⁶O₂ (between 9% and 13%). We also detected seven other oxidized Met on these GH7s, four were uniquely on the GH7 A, one on the GH7 B, and two were in a position conserved in GH7 A and B. Since we had assessed the overall level of Pro oxidation, we quantified the percentage of oxidation of individual Pro, considering only those labeled with ¹⁸O (Dataset 4). GH7 A had four oxidized Pro, two of which were in positions conserved into the GH7 B (Fig. 5A). Interestingly, we found that oxidation of Met and/or Pro occurred on homologs of GH16_2, PL35, proteases and GH152 (Fig. 4; Fig. S2). This represented 11 proteins among the 33 proteins identified having oxidized residues (28 with Met with or without Pro and 5 with Pro only) (Fig. 4; Fig. S2; Datasets 3; 4). Moreover, 19 out of the 33 identified proteins were oxidized on multiple residues, from two to 10. A GH152 was oxidized on 8 Pro, while the GH7 A was the protein with the highest number of oxidation sites (10), including six Met and four Pro (Fig. 4; Fig. 5; Fig. S2; Datasets 3; 4). Finally, several oxidation sites were located close to each other in the protein sequences, forming “hotspots” like the Pro343 and Met345, and three other Pro of the GH7 A (Fig. 5A). In total, we counted 24 occurrences of sites containing two oxidized residues in the protein sequences (Fig. 5B). Analysis of a tridimensional model of GH7 A revealed that oxidized residues distant on the sequence may be close on the structure, as the Met130 and Met160, the Met153 in the vicinity of three Pro, and the Met286 clustered with the Pro343 and Met345 (Fig. 5C). Altogether, these results indicate that most of the oxidation affects a relatively small set of CAZymes that can be oxidized at multiple positions, with some hotspots. Moreover, the fact that oxidized residues were found at similar positions within the homologs of P. cinnabarinus secreted proteins suggests that these residues have been conserved to fulfill antioxidant or regulatory roles.
A

<table><thead><tr><th></th><th></th><th>130</th><th>139</th><th>153</th><th>169</th><th></th></tr></thead><tbody><tr><td>A</td><td>A0A060SFM6</td><td>127</td><td>V Y L M D S S D Q K Y Q I F N L K N Q E F T F D V D M S K L P C G L N G A L Y F V E M D A D G G V S</td><td>176</td><td></td></tr><tr><td>B</td><td>A0A060ST00</td><td>127</td><td>V Y L L S E D D T T Y E M F N L K N Q E F T F D V D M S T L P C G L N G A L Y F V E M D A D G G K S</td><td>176</td><td></td></tr><tr><td>C</td><td>A0A060SMU8</td><td>127</td><td>V Y L M G T D D A S Y Q L F N L K N Q E F T F D V D M S N L P C G L N G A L Y F V E M D A D G G K S</td><td>176</td><td></td></tr></tbody></table>

<table><thead><tr><th></th><th></th><th>286/287</th><th>343/344</th><th>345/346</th><th></th></tr></thead><tbody><tr><td>A</td><td>A0A060SFM6</td><td>281</td><td>F N S W R M G D Q T F L G P G K T V N</td><td>299</td><td>338</td></tr><tr><td>B</td><td>A0A060ST00</td><td>282</td><td>F N S W R M G D Q T F L G S G K T V D</td><td>300</td><td>339</td></tr><tr><td>C</td><td>A0A060SMU8</td><td>281</td><td>F N S F R M G N Q T F L G P G K I V N</td><td>299</td><td>338</td></tr></tbody></table>

<table><thead><tr><th></th><th></th><th>378</th><th>422</th><th>424</th><th>430/431</th><th></th></tr></thead><tbody><tr><td>A</td><td>A0A060SFM6</td><td>366</td><td>T N S F E K L G G L K Q M G A S F D K G</td><td>385</td><td>420</td></tr><tr><td>B</td><td>A0A060ST00</td><td>367</td><td>T N T F E S L G G L K Q M G N A F Q Q G</td><td>386</td><td>421</td></tr><tr><td>C</td><td>A0A060SMU8</td><td>366</td><td>T N T F E Q L G G L Q Q M G N S F Q K G</td><td>385</td><td>420</td></tr></tbody></table>

B

<figure><img src="image_10.png" /></figure>

C

<figure><img src="image_11.png" /><figcaption>**Figure 5.** Partial sequence alignment of GH7, numbers of oxidation sites and hotspots in identified secreted proteins and tridimensional model of GH7. A) Oxidized Met and Pro residues are on *blue* and *orange* background, respectively. *Dark* and *light grey* backgrounds represent strictly conserved and similar amino acids, respectively. Position and percentage of oxidation of the residues are indicated above the sequence. For Pro, the percentage presented correspond to the sum of all oxidized forms, either ¹⁶O or ¹⁸O at day 3, excepted the one indicated with 'D5', corresponding to Pro oxidation at day 5. Underlined sequences represent peptides detected by LC-MS/MS. Accession numbers are on the left of the sequences. B) Values correspond to the total number of oxidizable Met and Pro sites found per protein. Hotspots correspond to the number of times that two oxidizable sites were located within a window of ten amino acids on the protein sequence. All data are presented in **Datasets 3 and 4**. C) 3D model of the GH7 A0A060SFM6 downloaded from Alphafold database (62). The position of the cellonoanose substrate (in *blue*) was modelized using the experimentally determined structure of *Trichoderma reesei Cel7A* (PDB #4C4C). Met and Pro found oxidized are represented as *cyan* and *orange* sticks, respectively. Catalytic residues are represented as *red* sticks. Percentage of oxidation at day 3 are in brackets. Some parts of the protein were rendered transparent for clarity.</figcaption></figure>
368

Conservation suggests a list of residues with antioxidant or regulatory roles.

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

To determine whether oxidized residues found in *P. cinnabarinus* proteins were widely conserved, we searched for homologs of the 33 proteins having oxidized residues in homologs from other fungi and quantified the conservation of the 89 oxidized residues. Our hypothesis was that oxidizable residues playing antioxidant or regulatory roles are more likely to be conserved among distantly related organisms, as shown for Met of actin or of the ffh/SRP proteins (63–65). To avoid overrepresentation of sequences coming from closely related organisms, the search was made in 120 representative fungal species, covering the nine fungal phyla (Dataset 5). Overall, the conservation of Met or Pro ranged from 1% to 100%, with a mean of 49%. Considering only residues with conservation values above 75%, and found in more than 80 sequences, we obtained a list of nine Met and nine Pro at conserved positions in 14 secreted proteins (Table 1). The GH7 A has four conserved Met, among which Met 169 conserved in 99% of the homologs, and a conserved Pro. Other Met are conserved in the GH7 B and C, and in two GH16_2, one GH18, one AA3_2 and a peptidase M36, present in all fungi (Table 1; Dataset 5). Regarding Pro, three are conserved in all homologs, at positions 614, 143 and 441 of a GH16_2, a GH28 and a laccase, respectively. Conserved Pro were also found in a AA3_2, an unknown protein and a GH152 (Table 1; Dataset 5). These results allowed identification of proteins with oxidizable Met and Pro residues strongly conserved across the fungal phyla.
<caption>Table 1. *P. cinnabarinus* secreted proteins with oxidized residues conserved across fungal proteins.</caption>

Only proteins for which the number of identified homolog sequences identified was higher than 80 were considered. The complete list of identified secreted proteins is presented in **Dataset 5**.

<table><thead><tr><th>Accession Uniprot (JGI)</th><th>Description</th><th>Repartition (Nb. of sequences)</th><th>Residue</th><th>Conservation (%)</th></tr></thead><tbody><tr><td rowspan="5">A0A060SFM6 (8640)</td><td rowspan="5">GH7</td><td rowspan="5">Dikarya (98)</td><td>Met 130</td><td>80</td></tr><tr><td>Met 169</td><td>99</td></tr><tr><td>Met 286<sup>a</sup></td><td>76</td></tr><tr><td>Met 378</td><td>83</td></tr><tr><td>Pro 430<sup>b</sup></td><td>99</td></tr><tr><td rowspan="2">A0A060ST00 (6809)</td><td rowspan="2">GH7</td><td rowspan="2">Dikarya (98)</td><td>Met 287<sup>a</sup></td><td>76</td></tr><tr><td>Pro 431<sup>b</sup></td><td>99</td></tr><tr><td>A0A060SMU8 (5845)</td><td>GH7</td><td>Dikarya (98)</td><td>Met 286<sup>a</sup></td><td>76</td></tr><tr><td>A0A060SISO (301)</td><td>GH16_2</td><td>Basidiomycota (100)</td><td>Met 615</td><td>92</td></tr><tr><td>A0A060S933 (3097)</td><td>GH16_2</td><td>Basidiomycota (91)</td><td>Pro 614</td><td>100</td></tr><tr><td>A0A060SC99 (6004)</td><td>GH18</td><td>Dikarya, Mucor., Zoo. (99)</td><td>Met 583</td><td>83</td></tr><tr><td>A0A060S4J1 (1984)</td><td>AA3_2</td><td>Agaricomycetes (100)</td><td>Met 342</td><td>86</td></tr><tr><td>A0A060SF78 (8374)</td><td>Pep. M36</td><td>All fungi (100)</td><td>Met 124</td><td>83</td></tr><tr><td>A0A060S2S5 (8001)</td><td>GH28</td><td>Dikarya (99)</td><td>Met 387</td><td>81</td></tr><tr><td>Q9UVQ2 (8672)</td><td>Laccase</td><td>Basidiomycota (100)</td><td>Pro 143</td><td>100</td></tr><tr><td>A0A060SC37 (3147)</td><td>AA3_2</td><td>Dikarya (98)</td><td>Pro 284</td><td>96</td></tr><tr><td>A0A060S4R6 (3419)</td><td>Unknown</td><td>Dikarya, Mucor., Chytridio., Neo. (100)</td><td>Pro 441</td><td>100</td></tr><tr><td>A0A060SF67 (3180)</td><td>GH152</td><td>Dikarya, Mucoromycota (99)</td><td>Pro 594</td><td>97</td></tr><tr><td>A0A060SY89 (5455)</td><td>GH152</td><td>Dikarya, Mucor., Chytridio. (99)</td><td>Pro 170</td><td>83</td></tr><tr><td></td><td></td><td></td><td>Pro 184<sup>c</sup></td><td>94</td></tr><tr><td></td><td></td><td></td><td>Pro 200<sup>c</sup></td><td>96</td></tr></tbody></table>

<footnote><sup>a, b, c</sup> Conserved positions between the *P. cinnabarinus* homologs. *Mucor*, *Mucoromycota*; *Zoo*, *Zoopagomycota*, *Chytridio*, *Chytridiomycota*, *Neo*, *Neocallimastigomycota*.</footnote>
392 **Discussion**

393 Taking a white-rot wood decayer as model, we showed that fungal secreted proteins have a
394 strongly reduced Met content (~1.4%) compared to the non-secreted (~2.1%) and that this feature is
395 not conserved in metazoans and plants. This decreased Met content suggests that protection of
396 proteins secreted by fungi is achieved by avoiding oxidation, in line with the previously proposed
397 mechanism (16). Interestingly, these data contrast with those obtained from animal studies which
398 showed that proteins encoded by the mitochondrial genome may contain up to 6-10% Met. This high
399 content could act as an endogenous antioxidant with the exposed Met scavenging ROS which are then
400 removed by methionine sulfoxide reductases in a cycle of oxidation and reduction (66). A similar
401 mechanism was proposed in bacteria (67, 68). The absence of external system of MetO reduction
402 preclude the use of such mechanism with fungal secreted proteins (24). This protection is most likely
403 accompanied by the secreted catalase we found (Fig. 3), in line with previous assumptions (16, 69–71).
404 The secretion of a catalase by *P. cinnabarinus* was unexpected, as it has not been previously detected
405 (41, 42), nor has extracellular activity been measured for the white-rot *Phanerochaete chrysosporium*
406 (72). However, searching for secreted catalase homologs, we found that it is widespread in
407 basidiomycota, and particularly in white-rot fungi (Table S5), contrary to previous phylogenetic
408 analysis (73). Lack of detection of the protein or its activity may be due to specific expression
409 conditions. In white-rot, it is surprising that a catalase was expressed along with H₂O₂ producing and
410 consuming enzymes. However, catalases have Km values in the mM range (70), and their action could
411 result in lowering the H₂O₂ concentration below a levels that is damaging to proteins, but allows the
412 activity of H₂O₂-driven oxidoreductases.

413 Using H₂¹⁸O₂-based proteomics, we identified oxidizable Met and highlighted that CAZymes
414 were the most oxidized secreted proteins during wood-decay, and that Met oxidation could occur on
415 several residues, sometimes clustered in hotspots. Clustering was also observed in bacterial proteins
416 (74), suggesting a universal phenomenon. The blocking strategy we used is based on a saturation of
reduced Met with <sup>18</sup>O. However, saturation was difficult to achieve, with only 4 Met saturated with <sup>18</sup>O in the day 3 secretome (Fig. 4). An initial denaturation step would have probably allowed H<sub>2</sub><sup>18</sup>O<sub>2</sub> to access the Met buried in the protein core (28). However, as our goal was to identify secreted proteins with potential oxidizable Met we chose not to denature them. Our idea was that an oxidizable Met should be accessible to an oxidant, either natural or H<sub>2</sub><sup>18</sup>O<sub>2</sub>. The results at day 3 argue for the accessibility of the Met to H<sub>2</sub><sup>18</sup>O<sub>2</sub>, as the percentage of <sup>18</sup>O was higher than that of <sup>16</sup>O for 43 Met out of 46. As artifactual <sup>16</sup>O-oxidation cannot be ruled out, the percentage of <sup>16</sup>O was necessarily lower or equal to those measured. In our study, the mean percentage of <sup>16</sup>O was 18%, while in studies on animals using similar approaches, it was ~4% (27, 28), arguing for potential artifactual oxidation. However, while intracellular proteins are protected by methionine sulfoxide reductases and ROS scavengers, the MetO in secreted proteins cannot be reduced, and no H<sub>2</sub>O<sub>2</sub>-consuming was detected in the secretome at day 3. Thus, to find high levels of MetO in fungal secreted proteins is expected. Interestingly, nine Met were oxidized as <sup>16</sup>O sulfone among which five were also oxidized as Met<sup>18</sup>O<sub>2</sub> (Fig. 4), confirming their propensity to form sulfone during wood decomposition.

Some of the Met we found oxidized in *P. cinnabarinus* are conserved throughout the fungal kingdom, suggesting potentially important roles (Table 5). They could act as antioxidants, as for the GH7 whose Met and Pro are located at the surface of the proteins and in the substrate cleft (Fig. 5C), potentially preventing critical residues from being oxidized, as proposed for a glutamine synthetase (23). Oxidizable Met could also have a functional role altering enzyme activity, like for a GH18 in which a conserved oxidized Met is located near the catalytic residue (Fig. S3), as shown for a GH27 galactosidase of *T. reesei* (25). In conclusion, our findings highlight a common mechanism in fungi preventing oxidation of secreted proteins and, pinpoint redox-regulated CAZymes to be further characterized.

440
441 Acknowledgments

442 The authors kindly thank the CIRM-CF for providing the *P. cinnabarinus* CIRM-BRFM145 strain, Dan
443 Cullen (Forest Product Laboratory, USDA, Madison, WI, USA) for providing *Populus tremuloides* wood,
444 and Marie-Noëlle Rosso (Aix Marseille Univ., INRAE, Biodiversité et Biotechnologies Fongiques) for
445 critical reading of the manuscript. This work received support from the French government under the
446 France 2030 investment plan, as part of the Initiative d'Excellence d'Aix-Marseille Université —IM2B
447 (AMX-19-IET-006) for L.M. and L.T. – A*MIDEX* (AMX-21-PEP-028) for L.T. The authors would like to
448 thank the NovoNordisk foundation (OxyMiST project, grant number NNF20OC0059697).

449

450 CRediT author statement

451 **Lise Molinelli**: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data
452 Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, **Elodie Drula**: Software,
453 Methodology, Formal analysis, Investigation, Data Curation, **Jean-Charles Gaillard**: Formal analysis,
454 Investigation, **David Navarro**: Investigation, Resources, **Jean Armengaud**: Formal analysis, Resources,
455 Data Curation, Writing - Review & Editing, **Jean-Guy Berrin**: Resources, Writing - Review & Editing,
456 Funding acquisition, **Thierry Tron**: Conceptualization, Resources, Writing - Review & Editing,
457 Supervision, Funding acquisition, **Lionel Tarrago**: Conceptualization, Methodology, Software,
458 Validation, Formal analysis, Investigation, Resources, Data Curation, Writing - Original Draft, Writing -
459 Review & Editing, Visualization, Supervision, Funding acquisition.

460
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