# Participation of a cysteine tetrad in the recycling mechanism of methionine sulfoxide reductase A from radiation-tolerant Deinococcus bacteria

Pascal Rey, Nicolas Rouhier, Chloé Carassus, Arjan de Groot, Laurence Blanchard

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Pascal Rey, Nicolas Rouhier, Chloé Carassus, Arjan de Groot, Laurence Blanchard. Participation of a cysteine tetrad in the recycling mechanism of methionine sulfoxide reductase A from radiation-tolerant Deinococcus bacteria. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2025, 1873 (3), pp.141063. 10.1016/j.bbvap.2025.141063. hal-05011335

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# Participation of a cysteine tetrad in the recycling mechanism of methionine sulfoxide reductase A from radiation-tolerant *Deinococcus* bacteria

Pascal Rey<sup>a</sup>, Nicolas Rouhier<sup>b</sup>, Chloé Carassus<sup>a,c</sup>, Arjan de Groot<sup>c,*</sup>, Laurence Blanchard<sup>c,*</sup>

## ARTICLE INFO

### Keywords:

Methionine sulfoxide reductase A
Redox-active cysteines
Disulfide isomerization
Thioredoxin
Deinococcus

## ABSTRACT

Methionine oxidation leads to the formation of methionine sulfoxide (MetO), which is reduced back to Met by methionine sulfoxide reductases (Msrs). The catalytic mechanism used by A-type Msr (MsrA) for MetO reduction requires a catalytic cysteine (Cys), which is converted to a sulfenic acid. In general, two resolving Cys are required for the regeneration of the catalytic Cys forming two consecutive disulfide bridges, the last one being efficiently reduced by thioredoxin (Trx). Here, we performed the biochemical characterization of MsrA from *Deinococcus deserti*. It possesses four Cys, two present in the active site motif (18 and 21) and two distal ones (53 and 163). We produced MsrA variants mutated for these cysteines and analyzed their capacity to reduce MetO in the presence of the NADPH-Trx reductase/Trx system, their ability to form heterodimers with Trxs, and their redox status after incubation with MetO. We show that all four Cys are involved in the regeneration process of enzyme activity by Trx. After MetO reduction by Cys18, a first disulfide bridge is formed with Cys21. A second disulfide involving Cys21 with either Cys53 or Cys163 is reduced by Trx, and a third Cys53-Cys163 disulfide can be formed and also reduced by Trx. These findings highlighting for the first time the involvement of a Cys tetrad in the catalytic and regeneration mechanisms for a MsrA are placed in a structural context by performing 3D modelling and discussed in relation to the known recycling mechanisms involving a Cys triad.

## 1. Introduction

*Deinococcus* bacteria exhibit a remarkable tolerance to extreme environmental stress conditions, such as radiation and desiccation, which impair cell redox homeostasis and lead to massive DNA damage [1,2]. This outstanding survival capacity involves a complex array of mechanisms protecting and/or repairing DNA and proteins [3–5]. Living organisms possess various thiol-dependent antioxidant systems including low-molecular-weight compounds such as bacillithiol, which is present in *Deinococcus* bacteria [4,6], and enzymes named thiol reductases, which harbor catalytic Cys that control the redox status of sulfur-containing residues in target proteins. The thioredoxin (Trx) system, in which NADPH provides electrons via NADPH-dependent Trx reductase (TrxR), allows disulfide bond reduction in many types of partners including peroxiredoxins (Prxs) and methionine sulfoxide reductases (Msrs), which scavenge peroxides and preserve protein redox status, respectively [7,8]. Trxs maintain the cell redox homeostasis upon diseases, environmental constraints or aging, through the modulation of

redox post-translational modifications and the prevention and repair of oxidative damage [9].

Methionine (Met) oxidation leads to the formation of Met sulfoxide (MetO) and Met sulfone (MetO₂), the latter modification being irreversible [10]. The S- and R-MetO diastereoisomers are specifically reduced back to Met by Msrs A and B, respectively. Both enzymes possess a very similar thiol-based redox chemistry [11] although having little sequence homology. MsrAs typically reduce peptide-bound and free MetO forms, while, depending on the organisms, MrBs reduce both or only efficiently the peptide-bound form [12–15]. Msr activity proceeds *via* a catalytic mechanism involving three main steps [16]: i) formation of a sulfenic acid intermediate on the catalytic Cys following MetO reduction; ii) formation of an intramolecular disulfide bond with a resolving Cys, possibly followed by disulfide isomerization involving an additional resolving Cys; iii) disulfide bond reduction by Trx *via* dithiol-disulfide exchange. In addition to a role in the control of protein redox status, protein activity, and repair of damaged proteins as recently shown for the RecA DNA repair protein [17], Msrs could fulfill a direct

<footnote><sup>a</sup> Aix Marseille Univ, CEA, CNRS, BIAM, Photosynthesis & Environment (P&E) Team, Saint Paul-Lez-Durance F-13115, France</footnote>

<footnote><sup>b</sup> Université de Lorraine, Inrae, IAM, F-54000 Nancy, France</footnote>

<footnote><sup>c</sup> Aix Marseille Univ, CEA, CNRS, BIAM, Molecular and Environmental Microbiology (MEM) Team, Saint Paul-Lez-Durance F-13115, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: nicolaas.degroot@cea.fr (A. de Groot), laurence.blanchard@cea.fr (L. Blanchard).</footnote>
antioxidant function by recycling oxidized Met [18]. Of note, there is growing evidence that Msrs participate in signaling functions in all organisms [19–22].

In radiation-tolerant *Deinococcus* bacteria, the control of Met redox homeostasis has only recently been explored. By comparing *Escherichia coli* and *Deinococcus radiodurans* proteomes following ionizing radiation (1000 Gy), Bruckbauer et al. [23] reported a much higher level of MetO formation in *E. coli* than in *D. radiodurans*, suggesting the presence of an efficient Mr repair system and/or of an antioxidant system limiting the formation of protein-bound MetO in the latter species. Upregulation of *Deinococcus* MsrA and MsrB expression has been reported upon oxidative stress conditions [24–27], as well as decreased resistance to oxidative stress for a *msrA* deletion mutant [27], further arguing for the participation of the Mr repair system in the remarkable tolerance of these bacteria to extreme conditions.

*Deinococcus* genomes harbor one *msrA* gene and one *msrB* gene [5,28]. *Deinococcus* MsrbS belong to the 2-Cys type, the activity of which is regenerated by Trx through the reduction of the intramolecular disulfide bond formed between the two Cys [29]. *Deinococcus* MsrAs possess four highly conserved Cys residues but their involvement in the catalytic act has never been explored and cannot be easily predicted based on previous studies [5]. In most previously characterized MsrAs, the catalytic Cys, which attacks and reduces MetO, is located in the conserved active site motif GCFW[G/S] [30–34]. The Trx-dependent recycling mechanism of MsrAs harboring such single-Cys-containing active site motif involves the participation of two other Cys in the C-terminal part and the formation of two consecutive disulfide bonds as described for *E. coli* and plant enzymes [34,35]. MsrAs from *Deinococcus* and some other species harbor an active site motif containing two Cys (GCFWC). Much less is known regarding the regeneration of this MsrA type by Trx, which could either reduce the disulfide bridge formed between both active site Cys [36] or another bridge involving a distal Cys following isomerization [37].

Here, we investigated the regeneration mechanism of *Deinococcus deserti* MsrA (DdMsrA), a 184-residue protein possessing four conserved Cys at positions 18, 21, 53 and 163, with Cys18 and 21 being part of the active site motif. We have expressed and purified the recombinant DdMsrA protein as well as variants in which the cysteines have been substituted by serines, and analyzed their ability to interact with the various Trxs present in this species and to reduce MetO in the presence of the NADPH-Trx reductase/Trx system. Based on our experimental data and 3D structure modelling, we propose a catalytic mechanism involving all four Cys with first the formation of a disulfide bridge between both Cys of the active site motif following MetO reduction, and then two consecutive isomerization steps involving the two distal Cys residues.

## 2. Materials and methods

### 2.1. Bacterial strain, plasmids and growth conditions

The bacterial strain *E. coli* BL21(DE3) (Invitrogen) was used to produce all recombinant proteins and Cys mutated variants. All plasmids used in this study are listed in Table S1. *E. coli* was grown with shaking (130 rpm) in lysogeny broth medium (LB) at 37 °C unless otherwise stated. Kanamycin was added to the growth media at 50 µg/ml. Plasmids for expressing proteins containing an N-terminal polyhistidine-tag were synthesized by GENEWIZ from Azenta Life Sciences. Briefly *D. deserti* msrA (locus tag Deide_10980), *trxR* (Deide_05800), *trxA* (Trx1, Deide_18600), *trxC* (Trx2, Deide_01140), *trx-like 1* (Trx-L1, Deide_06390), and *trx-L2* (Deide_13741) genes were synthesized as codon-optimized versions for expression in *E. coli* and cloned in pET-TEV vector [38] using *NdeI* and *XhoI* restriction sites. For *msrA*, the sequence encoding WP_041227141.1 (NCBI Reference Sequence) with corrected N-terminus was cloned. All Cys mutated variants of MsrA, Trx1 and Trx2 (Table S1) were also generated by GENEWIZ from Azenta Life Sciences.

### 2.2. Protein production, purification and electrospray ionization mass spectrometry

*Deinococcus deserti* MsrA, TrxR, Trx1, Trx2, Trx-L1, Trx-L2 proteins and Cys-to-Ser variants of MsrA, Trx1 and Trx2 were produced in *E. coli* BL21 (DE3) from the pET-TEV plasmid as N-terminal His-tagged fusion proteins (with the TEV protease cleavage site located directly after the His-tag). For expression of all proteins, *E. coli* BL21 (DE3) cells freshly transformed with the respective plasmids were grown at 37 °C overnight to saturation in 10 ml of LB medium containing kanamycin. This pre-culture was then diluted in 1 l of LB medium with kanamycin (in a 3 l flask) and grown at 37 °C, 130 rpm to an OD$_{600}$ of 0.6–0.7, before adding IPTG at a final concentration of 0.5 mM and incubation was continued at 17 °C for 17 h. The induced cells were harvested by centrifugation (6000 g, 15 min, 4 °C). The cell pellets were resuspended in 20 ml of Buffer 1 (500 mM NaCl, 10 mM Na$_2$HPO$_4$, 1.8 mM KH$_2$PO$_4$, pH 7.4), and then frozen at –20 °C. For purification, 20 ml of cell suspensions (in Buffer 1) containing the recombinant fusion proteins were thawed on ice and 100 µl of 10 mg/ml DNase I [DNase from bovine pancreas in 50 mM MgCl$_2$ (Sigma)] and 25 µl of anti-protease cocktail (Sigma P8849) were added. All purification steps were done at 4 °C. Cells were broken by a cell disruptor (One Shot Model, Constant Cell Disruption Systems) at 2 kbar and the soluble extracts were then recovered after centrifugation at 10,000 g for 20 min at 4 °C and ultracentrifugation at 150,000 g for 45 min at 4 °C. The supernatant was injected at a speed of 1 ml/min onto HisTrap™ HP columns (1 ml) (Cytiva), previously equilibrated in Buffer 1 supplemented with 20 mM imidazole. A step gradient of imidazole (20, 40 and 500 mM in Buffer 1) was used for elution (20 ml was used for each fraction except for the 500 mM fraction, for which only 4 ml was used). Eluted fractions were analyzed by SDS-PAGE to verify the presence and purity of the proteins. All proteins were recovered in the 500 mM imidazole fraction. Removal of imidazole from these fractions was done on a PD-10 desalting column (Cytiva) equilibrated with Buffer 1. To remove the polyhistidine tag, purified proteins at a concentration of 1 mg/ml were incubated with polyhistidine-tagged TEV protease overnight at room temperature (TEV protease:tagged protein with TEV cleavage site, mass ratio of 1:80). The cleavage reactions were then loaded on nickel affinity columns (HisTrap™ HP columns (1 ml) (Cytiva)) equilibrated in buffer 1 supplemented with 20 mM imidazole to separate untagged proteins (in the flow-through fractions) from non-cleaved fusion proteins, cleaved His-tag and TEV protease (all bound to the column). Purified untagged proteins were concentrated using an Amicon Ultra-15 Centrifugal filter unit (MWCO 10,000 or 3000, Millipore). The buffer of all protein samples was then changed to 50 mM potassium phosphate buffer pH 7.0, 50 mM NaCl, using PD-10 desalting columns (Cytiva). Protein concentrations were determined using a NanoVue spectrophotometer. SDS-PAGE (Novex NuPAGE 10 % Bis-Tris Gels, migration in 1xNuPAGE MES SDS running buffer (Invitrogen) for 1 h at 130 V) was used to follow each purification step and to verify the purity of the proteins, which were visualized by staining with Imperial protein stain (Pierce). The molecular weight marker proteins used are the SeeBlue Plus2 Prestained standard (Invitrogen). The mass of all purified proteins (MsrA, TrxR, Trx1, Trx2, Trx-L1, Trx-L2 and Cys-to-Ser variants of MsrA, Trx1 and Trx2) was verified by electrospray ionization mass spectrometry performed on a MicroTOF-Q (Bruker, Wissembourg, France) with an electrospray ionization source as described previously [39].

### 2.3. Biochemical and enzymatic assays

The reduction of insulin disulfide bridges by Trxs was assayed as described by Holmgren [40]. The assay mixture (1 ml) contained 50 mM potassium phosphate buffer pH 7.0, 2 mM EDTA, 1 mg/ml bovine pancreas insulin (Sigma) and 5 µM *D. deserti* Trx (DdTrx) or DdTrx-like proteins. The reaction was performed at room temperature and started by adding DTT at 0.67 mM (final concentration). The reduction of
insulin disulfide bridges was measured by the increase in turbidity at 650 nm due to protein precipitation. Reduction of insulin by DTT and by *E. coli* Trx1 (Sigma) were used as negative and positive controls, respectively.

The methionine sulfoxide reductase activity of DdMsrA and Cys-to-Ser variants was measured following NADPH oxidation at 340 nm in the presence of the DdTrx reducing system in 50 mM Tris pH 8.0, 1 mM EDTA and using as-purified DdMsrA. The reaction assay contained 200 µM NADPH (Roche), 2 µM *D. deserti* NADPH-dependent Trx reductase (DdTrxR), 0.1–10 µM of DdTrx1 or DdTrx2, 10 mM MetO (Sigma) and 5 µM DdMsrA. MsrA was added last after 30 s of incubation of the other compounds allowing reduction of Trx. The reaction was carried out at 25 °C in a 1 ml Quartz micro-cuvette, and the absorption curves were recorded using a Cary 50 spectrophotometer (Varian). The steady-state kinetic parameters were calculated from the slope due to NADPH consumption at varying Trx concentrations and MetO saturation (10 mM). The data were acquired from at least three independent measurements for each Trx concentration. The background consumption measured in the absence of DdMsrA was subtracted and calculations were performed considering that one mole of oxidized NADPH corresponds to one mole of MetO reduced or Met formed.

## 2.4. Analysis of the formation of *MsrA-Trx* heterodimers and of the redox status of *MsrA* cysteines

Incubation assays were performed to visualize the formation of heterodimers between *D. deserti* MsrA and Cys-to-Ser mutated variants on the one hand, and *D. deserti* Trx1 and Trx2, mutated or not for the resolving cysteine on the other hand. MsrA and Trx were incubated at equal concentrations (5 to 10 µM) in 40 µl of 50 mM potassium phosphate buffer pH 7.0 at 25 °C for 1 h. Before addition of as-purified Trx, MsrA was first incubated in open tubes at room temperature for 1 h to promote oxidation. Proteins were then separated by SDS-PAGE in the presence or absence of DTT before staining with Coomassie blue Impurity™ Protein Stain (Thermo).

The effect of MetO on the migration pattern of DdMsrA and Cys-to-Ser variants was investigated by incubating 10 µM enzyme in the presence of 10 mM MetO in 40 µl of 50 mM potassium phosphate buffer pH 7.0 at 25 °C for 1 h, and subsequent migration in non-reducing SDS-PAGE and Coomassie blue staining.

Alkylation assays were carried out to determine the number of free thiols in DdMsrA and Cys-to-Ser variants. Control assays on fully reduced proteins were performed following pre-reduction using 1 mM Tris(2-carboxyethyl)phosphine hydrochloride, TCEP, before alkylation. To analyze the thiol content after MetO reduction, MsrA proteins (10 µM) were incubated in the presence or absence of 10 mM MetO in 40 µl of 50 mM Tris-HCl pH 8.0 at 25 °C for 1 h, and SDS was added to the mix at 1 % final concentration. After 20 min, mPEG-maleimide-2000 (Laysan Bio Arab, AL, USA) was added at 2.5 mM to the mixture, which was further incubated at 25 °C for 3 h. Then, the reaction mixture was added with reducing loading buffer for SDS-PAGE and Coomassie blue staining.

## 2.5. Multiple sequence alignment

Multiple sequence alignments of representative MsrA proteins with one-Cys and two-Cys active site motif were made with UniProt ClustalO [41]. UniProt or NCBI accession numbers of selected MsrA proteins are WP_041227141.1 (*D. deserti*), WP_051618834.1 (*D. radiodurans*), P54149 (*Bos taurus*), AAS46232.1 (*Populus trichocarpa* × *Populus deltoides*), P0A744 (*E. coli*), Q9JWM8 (*Neisseria meningitidis* PiIB), P9WJM5 (*Mycobacterium tuberculosis*), P54154 (*Bacillus subtilis*), Q9KAN8 (*Halalkalibacterium halodurans*), Q72UW2 (*Leptospira interrogans* MsrA1), Q72N60 (*Leptospira interrogans* MsrA2).

## 2.6. 3D protein structure modelling

The 3D structures of MsrAs from *D. deserti*, *L. interrogans*, and *B. subtilis* possessing a two-Cys active site motif were predicted using AlphaFold2 via the software ColabFold [42,43]. All parameters were kept to default values. The quality of each model was evaluated using the confidence measures pLDDT (predicted local distance difference test) that indicates the local accuracy, and PAE (predicted aligned error) that assesses the packing between domains or protein chains. The X-ray 3D structure of *E. coli* MsrA having a one-Cys active site motif was obtained from the protein data bank (PDB ID: 1FF3). Three-dimensional structure or model images (rank_1 model for each AlphaFold2 predicted 3D structure) were generated using PyMOL (PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

# 3. Results

## 3.1. Sequence features of *Deinococcus deserti* *MsrA*

Two MsrA types can be distinguished depending on the presence of one or two Cys in the active site motif, GCFW[G/S] or GCFWC, located in the N-terminal part of the protein [32,37]. Further diversity exists in the number of additional, distal Cys in both types. For instance, *M. tuberculosis* MsrA and the MsrA domain of *N. meningitidis* PiIB possess a GCFWG motif and one additional Cys (corresponding to Cys163 of DdMsrA), while MsrA from *B. subtilis* and *H. halodurans* possess a GCFWC motif and one (corresponding to Cys53 of DdMsrA) or no additional Cys, respectively (Fig. S1). Other MsrAs often contain several extra Cys, which are more or less conserved (Fig. S1). In fact, the position of extra Cys, some of which involved in the recycling of the MsrA catalytic Cys by allowing disulfide isomerization and subsequent reduction by Trx, greatly varies [34,35,37].

*Deinococcus* MsrAs harbor a two-Cys active site motif and two additional Cys residues. An amino acid sequence alignment with previously characterized MsrAs indicates that Cys18 of DdMsrA corresponds to the catalytic Cys (Fig. 1). The vicinal Cys (Cys21) could be a (first) resolving Cys as reported for the equivalent Cys in *L. interrogans* MsrAs [37] and suggested for *B. subtilis* MsrA (Fig. 1) [32]. The two distal Cys of DdMsrA (Cys53 and Cys163) are conserved in several MsrA sequences (Fig. 1, Fig. S1), but only the one equivalent to Cys163 was shown or proposed to function as a resolving Cys in previously studied MsrAs [31,32,34,35,37].

## 3.2. Reduction of *MetO* by DdMsrA in the presence of typical or atypical Trxs

*Deinococcus deserti* expresses two canonical Trxs, Trx1 and Trx2, the latter possessing an additional N-terminal domain, that should bind zinc, given the analogy with *E. coli* Trx2. In addition, it expresses two Trx-like proteins, Trx-L1 and Trx-L2 that are highly conserved in *Deinococcus* [5,28]. *D. deserti* MsrA, TrxR, Trx and Trx-like proteins and Cys-to-Ser variants of MsrA and Trxs were purified after expression in *E. coli* (Table S1; Fig. S2). We first analyzed the ability of DdTrx and DdTrx-like proteins to catalyze the reduction of insulin in the presence of DTT [40]. DTT alone led to almost no insulin precipitation before 40 min. In the presence of *E. coli* Trx, insulin precipitation occurred after 8 min, while in the presence of DdTrx1 and DdTrx2, earlier and faster precipitation was observed, after 4 and 6 min, respectively (Fig. 2A). When assays were carried out with DdTrx-L1 or DdTrx-L2, no reduction of insulin was observed (Fig. 2A). We then performed similar assays using Trxs mutated for their resolving Cys (Table S1), and observed that insulin reduction was abolished for both DdTrx1 C32S and DdTrx2 C67S (Fig. 2B). These data validate the disulfide reductase activity of both canonical DdTrxs.

The Trx-dependent methionine sulfoxide reductase activity of DdMsrA was measured spectrophotometrically by following NADPH
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Multiple alignment of MsrA proteins from *D. deserti* and three other bacteria. The demonstrated or proposed catalytic (cat) and resolving (res) Cys residues in MsrA proteins from *E. coli* (Ecol), *L. interrogans* (Lint) and *B. subtilis* (Bsub) are shown as white letters in black background and indicated with closed triangles below the alignment. Other Cys are shown as white letters in grey background. The four Cys (18, 21, 53, 163) in MsrA from *D. deserti* (Ddes) are indicated with open triangles above the alignment. *D. deserti* MsrA has 43 %, 47 % and 38 % identity with MsrA from *E. coli*, *L. interrogans* (MsrA1) and *B. subtilis*, respectively.</figcaption></figure>

oxidation at 340 nm in the presence of *D. deserti* NADPH-dependent Trx reductase (DdTrxR), various concentrations of DdTrx and DdTrx-L proteins and a saturating MetO concentration. Control assays performed in the absence of DdMsrA revealed very low and linear levels of NADPH oxidation, whereas adding DdMsrA (5 µM) with DdTrx1 or DdTrx2 in the range of 1 to 10 µM resulted in increasing NADPH consumption (Fig. 2C-D). These data indicate that DdMsrA can employ both canonical Trxs as physiological reductants. When the activity of Trxs lacking their resolving Cys was assayed, no NADPH consumption was observed, which is consistent with the reduction of DdMsrA via dithiol-disulfide exchange (Fig. 2C-D). Finally, no NADPH oxidation was noticed when assays were carried out using either DdTrx-L1 or DdTrx-L2 (Fig. S3), suggesting either that they cannot reduce MsrA and/or that they are not reduced by TrxR.

The kinetic parameters of DdMsrA measured in the presence of varying concentrations of DdTrx1 or DdTrx2 and at a saturating MetO concentration were calculated (Table 1). The apparent $K_M$ values determined under steady-state conditions are 2.1 and 1.3 µM for DdTrx1 and DdTrx2, respectively, and the catalytic efficiencies expressed as $k_{cat}/K_{M,Trx}$ are in the range of $100 \times 10^3 \text{ M}^{-1}\cdot\text{s}^{-1}$, ca. $90 \times 10^3$ and $150 \times 10^3$, respectively. These data indicate a slightly higher efficiency of DdTrx2 compared to DdTrx1 for the reduction of DdMsrA.

### 3.3. Identification of DdMsrA Cys residues involved in the catalytic mechanism

#### 3.3.1. Complex formation between DdMsrA and the two canonical DdTrxs

Mutation of the resolving Cys in Trxs leads to stabilization of disulfide-bridge heterodimers formed with their targets after the first reaction step [44]. To investigate the interaction between DdMsrA and Trx1, Trx2 or their respective Cys mutated variants, oxidized DdMsrA was incubated with as-purified Trx proteins in the absence of MetO.

Therefore, these experiments provide information on the intrinsic reactivity of thiol groups from both proteins and on the ability to form protein-protein complexes involving intermolecular disulfides when the mixtures are analyzed by SDS-PAGE under non-reducing conditions (Fig. 3). In the absence of MsrA, Trxs migrated essentially as monomers (Fig. 3A, lanes 2, 9), whereas covalent dimers were observed for Trx1 C32S and Trx2 C67S (in higher amounts for the former) (Fig. 3A, lanes 2–3, 9–10). These dimers were not observed in reducing conditions (Fig. S4A). For DdMsrA, several redox monomers and higher forms were observed (Fig. 3A, lane 6). The absence of the latter in reducing SDS-PAGE indicated that these forms are dimers resulting from disulfide bridging of two monomers (Fig. S4A). Still, two persistent monomeric forms are observed in reducing SDS-PAGE (Figs. S2A and S4A), which are assumed to correspond to two different MsrA redox forms since a single species was detected by mass spectrometry. Incubation of DdMsrA in the presence of either Trx1 or Trx2 did not reveal any additional band compared to the respective proteins (except a relatively intense one of ca. 48 kDa with Trx1) (Fig. 3A, lanes 4, 7 compared to 2, 6, 9). In contrast, two additional bands approximately at 32 kDa and 55 kDa were observed following incubation of DdMsrA and Trx1 C32S, these bands being absent in reducing conditions (Fig. 3A and Fig. S4A, lanes 4–5). Similarly, an additional band at ca. 35 kDa was observed only in non-reducing conditions when mixing MsrA and Trx2 C67S (Fig. 3A and Fig. S4A, lanes 7–8). From the molecular weights of the respective proteins, the 32- and 35 kDa-bands likely correspond to MsrA-Trx heterodimers formed by one MsrA monomer and one Trx monomer, linked by a disulfide bridge. The 55-kDa additional band observed when mixing Trx1 C32S with MsrA could correspond to oligomers composed of one or two monomers of each redox partner.

Then, in order to decipher the mechanism by which DdMsrA activity is regenerated by Trxs, we aimed to identify the Cys residues among Cys21, Cys53 and Cys163 involved in the recycling process after MetO
A

<figure><img src="image_4.png" /></figure>

B

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

C

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

D

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Reduction by DdTrxs of insulin and of DdMsrA. (A) Insulin reduction by purified DdTrxs at 5 µM. DTT alone and *E. coli* Trx (EcTrx) at 5 µM were used as negative and positive controls, respectively. (B) Insulin reduction by purified wild-type (WT) or Cys-to-Ser mutated DdTrxs at 5 µM. The reaction was started by the addition of DTT (0.67 mM). (C) NADPH consumption in the presence of DdTrx1 and DdMsrA. (D) NADPH consumption in the presence of DdTrx2 and DdMsrA. NADPH consumption was measured spectrophotometrically. The assay mixture contained the Trx reducing system: 200 µM NADPH, 2 µM DdTrxR, varying concentrations (0.1, 1 and 10 µM) of WT or Cys-to-Ser mutated Trx, a saturating concentration of MetO (10 mM) and 5 µM DdMsrA. Control assays were carried out using 10 µM Trx without DdMsrA. The data shown are representative of at least three independent experiments performed in similar conditions.</figcaption></figure>

**Table 1**

Catalytic parameters of DdMsrA and of the C53S and C163S variants using DdTrx1 (A) and DdTrx2 (B) as electron donors. These parameters were determined under steady-state conditions by spectrophotometrically following NADPH consumption in the presence of Trx reducing system. The assay mixture contained 200 µM NADPH, 2 µM DdTrxR, varying concentrations (from 0.1 to 10 µM) of Trx1 or 5 µM of Trx2, a saturating concentration of MetO (10 mM) and 5 µM DdMsrA or Cys-to-Ser variants. Data are means ± S.D. of three values originating from independent experiments.

<table><thead><tr><th></th><th>k<sub>cat</sub> (s<sup>-1</sup>)</th><th>K<sub>M</sub> Trx1 (µM)</th><th>k<sub>cat</sub> / K<sub>M</sub> Trx1 (x 10<sup>3</sup> M<sup>-1</sup> s<sup>-1</sup>)</th></tr></thead><tbody><tr><td colspan="4"><strong>A</strong></td></tr><tr><td>DdMsrA</td><td>0.19 ± 0.02</td><td>2.1 ± 0.0</td><td>91 ± 10</td></tr><tr><td>DdMsrA C53S</td><td>0.28 ± 0.02</td><td>2.5 ± 0.1</td><td>112 ± 11</td></tr><tr><td>DdMsrA C163S</td><td>0.15 ± 0.12</td><td>1.8 ± 0.2</td><td>88 ± 16</td></tr><tr><td colspan="4"><strong>B</strong></td></tr><tr><td>DdMsrA</td><td>0.19 ± 0.02</td><td>1.3 ± 0.1</td><td>147 ± 2.0</td></tr><tr><td>DdMsrA C53S</td><td>0.18 ± 0.02</td><td>1.7 ± 0.0</td><td>105 ± 11</td></tr><tr><td>DdMsrA C163S</td><td>0.16 ± 0.03</td><td>2.5 ± 0.4</td><td>62 ± 3</td></tr></tbody></table>

reduction by the catalytic Cys18. We have produced and purified MsrA variants harboring one, two or three Cys mutated to Ser. Incubation experiments were then performed with all MsrA variants to compare their ability to form disulfide-bridged heterodimers with either Trx1

C32S (Fig. 3B) or Trx2 C67S (Fig. 3C).

The 32-kDa band observed in the MsrA-Trx1 C32S mix (Fig. 3A, lane 5) was still clearly present in all mixes containing single Cys-to-Ser MsrA variants, but was more abundant in mixtures containing MsrA C53S or MsrA C163S (even more for the former) (Fig. 3B, lanes 3–7). Notably, for the two other single mutants (MsrA C18S and C21S), the pattern was somewhat modified with the appearance of two close but distinct bands at the corresponding size (ca. 32 kDa), these bands being less abundant in the case of MsrA C18S (Fig. 3B, lanes 4–5). Very interestingly, the 32-kDa band corresponding to MsrA-Trx1 C32S heterodimer was still present in the mixture containing the double C18S-C21S MsrA variant (Fig. 3B, lane 8), but not in the reaction mix with the other double mutant (C53S-C163S) (Fig. 3B, lane 9). Of note, the 55-kDa oligomer band observed in the presence of DdMsrA was barely detected in incubation mixtures containing enzyme variants (Fig. 3B, lanes 4–9).

The 35-kDa band in the MsrA-Trx2 C67S mix (Fig. 3A, lane 8) was still detected in mixtures with single Cys-to-Ser MsrA variants. It was much more abundant in the mix containing MsrA C53S (Fig. 3C, lanes 3 and 6), faintly observed with MsrA C18S or C21S and barely detectable with MsrA C163S (Fig. 3C, lanes 4, 5, 7). In the case of MsrA variants with double mutations, the 35 kDa-band was only observed with MsrA C18S-C21S (Fig. 3C, lane 8).

Taken collectively, these data reveal the ability of DdTrx1 and DdTrx2 to form disulfide bridges with DdMsrA, via Cys53 or Cys163, since no heterodimer was observed when incubated with MsrA lacking
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 3.** *In vitro* analysis of the formation of heterodimeric complexes between DdTrxs and DdMsRA proteins and cysteine-mutated variants. (A) Formation of heterodimers between DdMsRA and DdTrxs mutated or not. (B) Formation of heterodimers between the DdTrx1 C32S variant, and DdMsRA mutated or not. (C) Formation of heterodimers between the DdTrx2 C67S variant, and DdMsRA mutated or not. Proteins (10 µM each) were incubated in phosphate buffer for 1 h at room temperature, and then separated in non-reducing SDS-PAGE conditions. Gels were stained using Coomassie blue. Asterisks indicate the appearance of additional bands corresponding to Trx-MsRA heterodimers. Lanes 1, molecular weight marker proteins (masses, in kDa, are indicated). WT, wild-type. The data shown are representative of three independent experiments performed in similar conditions.</figcaption></figure>

these two Cys residues, and harboring only Cys18 and Cys21. This implies that the Cys18-Cys21 disulfide is likely not reduced by Trx and that Cys53 and Cys163 are involved in disulfide isomerization and act in the Trx-mediated recycling process.

### 3.3.2. Roles of the cysteines of DdMsRA for the Trx-mediated MetO reduction

We then investigated MetO reduction by the MsRA variants in the presence of Trx1 or Trx2 using the NADPH-coupled spectrophotometric assay described above. Similar results were obtained with both Trxs (Fig. 4A-B). We first noticed that MsRA variants lacking either both Cys18 and Cys21 or both Cys53 and Cys163 did not exhibit any Trx-dependent MetO reductase activity, as well as the variant lacking Cys21, Cys53 and Cys163. This indicates that Trx can reduce neither the

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 4.** Reduction of MetO by DdMsRA and Cys-to-Ser variants in the presence of DdTrx1 (A) or DdTrx2 (B). NADPH consumption was measured spectrophotometrically and assays were carried out in the presence of the Trx reducing system. The assay mixture contained 200 µM NADPH, 2 µM DdTrxR, 10 µM Trx1 or 5 µM Trx2, a saturating concentration of MetO (10 mM) and 5 µM DdMsRA proteins. Control assays were carried out without DdMsRA. WT, wild-type. The data shown are representative of at least three independent measurements performed in similar conditions.</figcaption></figure>

sulfenic acid formed on Cys18, nor a Cys18-Cys21 disulfide.

Regarding single mutated variants, the activity of MsRA C18S was completely abolished in agreement with the assumption that Cys18 serves as the catalytic residue in the process of MetO reduction. A strong decrease in the Trx-dependent MetO reduction was observed for MsRA C21S with a 5- and 7-fold decrease in the initial velocities measured in the presence of Trx1 or Trx2, respectively, as compared to those measured with the non-mutated DdMsRA. Still, this indicates that in the context of this C21S variant, one of the remaining Cys is able to react with the sulfenylated Cys18. In contrast, the capacity of MsRA C53S and MsRA C163S to reduce MetO was not substantially altered in the presence of Trx1, with apparent $K_M$ values close to 2 µM and catalytic efficiencies in the order of $100 \times 10^3 M^{-1}.s^{-1}$. In the presence of DdTrx2, slightly lower catalytic efficiencies of MsRA C53S and MsRA C163S were measured, $105 \text{ and } 62 \times 10^3 M^{-1}.s^{-1}$, respectively, vs $147 \times 10^3 M^{-1}.s^{-1}$ for DdMsRA (Table 1).

Altogether, the data gained with these Trx-dependent MetO reduction tests confirm that Cys18 is the catalytic residue and reveal that the three other Cys are involved in the recycling process by Trx. The strongly reduced activity recorded with the MsRA C21S variant coupled with the inability of the MsRA C53S-C163S variant to be recycled by Trx suggest that Cys21 is the first attacking Cys of the sulfenic acid formed on Cys18 and that the Cys18-Cys21 cannot be reduced by Trx, possibly for accessibility reasons. Then, Cys53 or Cys163 would react with the Cys18-Cys21 disulfide. In these *in vitro* assays, both Trx1 and Trx2 are able to react with either of these newly formed disulfide bonds. The
question is whether Cys53 and Cys163 can replace each other in the respective protein variants or whether they act consecutively in the non-modified protein. The formation of a MsrA-Trx1/2 heterodimer using the C18S-C21S MsrA variant indicates that a Cys53-Cys163 disulfide can at least be formed and react with Trx, thus supporting the involvement of the four cysteine residues in the regeneration process.

### 3.4. Effect of MetO on DdMsrA migration in non-reducing conditions

We then investigated the effect of incubating DdMsrA and the Cys-mutated variants with a large excess of MetO (10 mM). Indeed, based on the structural modelling (see below) and previous studies performed on *Bos taurus* and *L. interrogans* MsrAs [31,37], it may be expected that the formation of intra- or intermolecular disulfide bridges promoted by the addition of MetO results in a relatively large conformational change thus altering the migration properties of the protein in non-reducing conditions. Consistent with this expectation, a strong and complete electrophoretic mobility shift towards lower molecular weights (from 23 kDa to *ca.* 17–18 kDa) was observed following incubation of DdMsrA with MetO compared to that of the untreated protein (Fig. 5, lanes 2–3). While no effect of MetO addition was noticed for the MsrA C18S variant, a similar effect was observed with the three other variants with a single mutated cysteine. However, the mobility shift is different from that of DdMsrA, very likely due to a different disulfide status (Fig. 5, lanes 4–11). Finally, no mobility shift was observed for the double and triple variants (Fig. S5A). In conclusion, initial oxidation of Cys18 is mandatory for the conformational change to occur and the formation of the Cys18-Cys21 disulfide is not responsible of the large conformational change, which requires a further reaction of Cys18-Cys21 disulfide with Cys53 or Cys163.

### 3.5. Thiol content in DdMsrA and Cys-mutated variants after incubation with MetO

We then examined the redox status of DdMsrA cysteines upon incubation with MetO by performing alkylation experiments using mPEG-maleimide-2000, which forms stable thioether bonds with thiol groups, allowing the detection of reduced Cys due to a 2-kDa size increase per free thiol. We first performed experiments on DdMsrA and Cys-mutated variants in the presence of the strong reducing agent TCEP, which is appropriate for Cys labeling with maleimides [45]. After alkylation and migration in reducing conditions, the major species for DdMsrA likely corresponds to a form with the four Cys alkylated, but the presence of other bands indicate that reduction was incomplete even when using

<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Effect of MetO on the electrophoretic properties in non-reducing conditions of DdMsrA and single Cys-to-Ser variants. Equal amounts (12 µg) of purified proteins were incubated or not with 10 mM MetO in phosphate buffer for 1 h and then separated in non-reducing SDS-PAGE conditions. Gels were stained using Coomassie blue. Lane 1, molecular weight marker proteins (masses, in kDa, are indicated). The data shown are representative of three independent experiments performed in similar conditions.</figcaption></figure>

TCEP (Fig. 6A). For the four variants with a single mutation, alkylation of the three Cys was observed but the reaction was still incomplete. For the C18S-C21S variant, it appeared that the two remaining Cys are predominantly alkylated. For the C53S-C163S variant, the pattern was less obvious to interpret, since there were indeed two higher bands, but these alkylated forms migrated faster compared to those observed with the C18S-C21S variant (Fig. 6A). We then performed alkylation assays following incubation with MetO to analyze the redox status of MsrA cysteines upon MetO reduction and subsequent modifications. Alkylation of as-purified DdMsrA also revealed the presence of four bands, but with a much higher proportion of the one-thiol free form, compared to the pattern observed following treatment with TCEP (Fig. 6A-B, lanes 2). Following incubation of DdMsrA with MetO, the non-alkylated form was the most abundant, the one-thiol free band was still present and the three others disappeared (Fig. 6B, lanes 2–3). Similarly, MetO and maleimide treatments led to substantial changes in the number of free thiols in C21S, C53S and C163S MsrA variants since the non-alkylated form was predominant for the three forms (Fig. 6B, lanes 4–9). Conversely, no noticeable change was observed in C18S, C18S-C21S and C53S-C163S DdMsrA variants treated for alkylation following oxidation by MetO (Fig. S5B). Taken collectively, these data corroborate the role of Cys18 as the catalytic one in the process of MetO reduction by DdMsrA, and indicate that the three other Cys undergo subsequent oxidation of their thiol group during the first steps of enzyme activity regeneration.

A

<figure><img src="image_14.png" /></figure>

B

<figure><img src="image_15.png" /><figcaption>**Fig. 6.** Alkylation pattern of DdMsrA or Cys-to-Ser variants. (A) Purified proteins were first pre-reduced using 1 mM TCEP for 1 h, and then subjected to alkylation by incubation in the presence of mPEG-maleimide-2000 for 3 h. (B) Equal amounts (10 µM) of purified proteins were first incubated or not with 10 mM MetO for 1 h, and then subjected to alkylation by incubation in the presence of mPEG-maleimide-2000 for 3 h. Proteins were separated in reducing SDS-PAGE conditions and gels were stained using Coomassie blue. The data shown are representative of three independent experiments performed in similar conditions.</figcaption></figure>
### 3.6. Structure modelling of DdMsrA and comparison with other MsrAs

The 3D structure of *D. deserti* MsrA predicted by AlphaFold2 with high confidence shows that it is folded into an alpha/beta roll containing a large proportion of coils as observed for the available X-ray 3D structure of *E. coli* MsrA, which harbors a one-Cys active site motif (Fig. 7, Fig. S6). Based on the results described above, the four Cys in the DdMsrA model (Fig. 7A) are colored according to their function of catalytic (orange), first resolving (blue) or second/third resolving Cys (green). These Cys are located relatively close to each other in the 3D model, but the distances between sulfur atoms support the experimental data that the formation of the successive intramolecular disulfide bridges upon MetO reduction involves conformational changes. The two Cys (18 and 21) of the active site motif are positioned at the beginning of an α-helix, the catalytic Cys18 showing a perfect superposition with the catalytic Cys51 of *E. coli* MsrA (Fig. 7, Fig. S6D). One of the second resolving Cys of DdMsrA, Cys53, superposes with *E. coli* MsrA Cys86, which, however, was found to have no role in catalysis [35]. In the superposed MsrA structures, the other second resolving Cys of DdMsrA, Cys163 (present in an α-helix), is located very close to the first resolving Cys198 (present in a coil) in *E. coli* MsrA (6 Å between main chain carbon). The sulfur of the first resolving Cys21 in DdMsrA is located at a distance of 13.4 Å and 8 Å from the sulfur of the two second resolving Cys, Cys53 and Cys163, respectively. The distance between the sulfur of Cys53 and Cys163 is 15.4 Å in the model with all Cys reduced. The AlphaFold2 generated 3D models of two other members of the two-Cys active site motif MsrAs (*L. interrogans* MsrA1 and *B. subtilis* MsrA) show the same fold and perfectly superpose with the DdMsrA model, with the same position of the two Cys of the active site motif. The only extra Cys (Cys49) of *B. subtilis* superposes perfectly with Cys53 of DdMsrA, and two of the three extra Cys (Cys57 and Cys165) of MsrA1 of *L. interrogans*

<figure><img src="image_16.png" /><figcaption>**Fig. 7.** 3D structures of MsrA highlighting the position of Cys residues. The 3D structures of MsrAs with a two-Cys active site motif from *D. deserti* (A), *L. interrogans* (C) and *B. subtilis* (D) have been predicted by AlphaFold2 for comparative purposes together with *E. coli* MsrA X-ray 3D structure (PDB ID: 1FF3) (B) used as a representative of MsrA with one-Cys active site motif. Of note, in EcMsrA, the catalytic Cys51 is replaced by S-(dimethylarsenic)cysteine. The 3D structures are colored according to secondary structure elements (α-helices in pink and β-strands in teal blue). The Cys residues are colored according to their demonstrated or proposed function in the MsrA catalytic mechanism, with the catalytic Cys in orange, the first resolving Cys in blue and second/third resolving Cys in green.</figcaption></figure>

superpose also perfectly with Cys53 and Cys163 of DdMsrA, respectively (Fig. 7, Fig. S6). Together, the two distal Cys in the DdMsrA 3D model are present at similar positions in the 3D structure or model of other MsrAs, but DdMsrA is the only prototype so far for which both Cys participate in the recycling process.

## 4. Discussion

### 4.1. Participation of DdTrxs and DdTrx-like proteins in DdMsrA recycling

MsrAs are ubiquitous enzymes fulfilling essential roles in the maintenance of protein redox homeostasis thanks to reducing power generally supplied by Trxs. Among the four Trx and Trx-like (Trx-L) proteins of *D. deserti*, only the two canonical Trxs, Trx1 and Trx2, are able to regenerate MsrA activity (Fig. 2; Fig. S3). For Trx-L1, which belongs to the TRP14/Clot class, these data are in agreement with those reported for the TRP14 homolog from poplar [46]. Based on the kinetic parameters measured in steady-state conditions, the calculated catalytic efficiency values of DdTrx1 and DdTrx2 towards DdMsrA are 90 and 150 × 10³ M⁻¹.s⁻¹, respectively (Table 1). These values are in the range of those reported for MsrA enzymes from various eukaryotes and prokaryotes, which stand from 20 to 500 × 10³ M⁻¹.s⁻¹ [32,34,37,47]. In *E. coli*, Trx2 was first proposed not to be involved in MsrA recycling because a mutant strain lacking Trx1 was unable to grow in the presence of MetO [48]. However, Jacob et al. [47] showed that EcTrx2 exhibits *in vitro* the ability to reduce EcMsrA and concluded that the phenotype of the mutant originated from a very low level of *trx2* expression (150-fold lower than that of *trx1*). In *D. deserti*, de Groot et al. [25] also noticed a similar differential expression pattern for the two *trx* genes in standard conditions. Of note, both genes are induced after exposure to H₂O₂ in *D. radiodurans* [49]. While we observed that the catalytic efficiency of DdTrx2 towards MsrA (150 × 10³ M⁻¹.s⁻¹) was higher than that of DdTrx1 (90 × 10³ M⁻¹.s⁻¹), Jacob et al. [47] reported a substantially lower catalytic efficiency for EcTrx2 towards EcMsrA, compared to that of EcTrx1, 70 and 500 × 10³ M⁻¹.s⁻¹, respectively. This difference might be related to the fact that *Deinococcus* and *E. coli* MsrAs are distinguished by different active sites and Cys number and repartition. This assumption is corroborated by the fact that a much lower initial velocity was recorded when using EcTrx1 to reduce DdMsrA instead of DdTrxs (Fig. 4, Fig. S3B).

### 4.2. Variability in MsrA regeneration mechanisms in relation with Cys number and position

MsrAs are classified in two main types [37] based on the presence of one or two Cys in their active site, GCFW[G/S/C]. Most MsrAs harbor other Cys and there is an important diversity in their number and position [32,34,37,50]. Although they exhibit quite divergent sequences (Fig. 1, Fig. S1), their structures are highly conserved (Fig. 7, Fig. S6). Plant MsrAs contain up to four Cys outside the active site, while three are present in *E. coli* or *B. taurus* sequences and two in *Deinococcus* enzymes. Of note, some MsrAs contain a lower number of Cys residues, like *H. halodurans* MsrA that has only two Cys positioned in the active site (Fig. S1), while others possess only one Cys (located in the active site) [51]. This diversity implies distinct recycling mechanisms. MsrAs having a single Cys are regenerated by the glutathione/glutaredoxin system [51,52]. For most other MsrAs, the Trx system represents the major or unique reduction system, as hypothesized for MsrAs harboring two Cys in the active site that form an intramolecular disulfide bridge [36], and reported for MsrAs having only one Cys in the active site, for which additional isomerization steps involving two distal resolving Cys are required [31,34,35]. MsrAs harboring two Cys in the active site, such as *Deinococcus* MsrAs, are specific to prokaryotes and found in Archaea and in some bacterial phyla, including Firmicutes and Spirochaetes (e.g., *L. interrogans*) [37]. Of note, most of them harbor one to three additional, distal Cys. Based on the properties of *L. interrogans* MsrA variants
lacking the second active site Cys, Sasoni et al. [37] recently proposed that the regeneration mechanism by Trx also involves a Cys triad, the two active site cysteines and a distal one (corresponding to Cys163 of DdMsrA) required for an isomerization step.

### 4.3. Disulfide exchange mechanisms in DdMsrA recycling

In the case of DdMsrA, we concluded from our experiments that the activity recycling by Trx also requires the participation of at least one distal cysteine. Indeed, when measuring MetO reduction in the presence of the Trx system, no activity was recorded for the DdMsrA protein harboring only the two Cys of the active site (Fig. 4). Strongly decreased activity was measured for the MsrA lacking Cys21 (Fig. 4), indicating that this residue is critical for the recycling process. The residual activity measured with this variant could be due to reduction by Trx of disulfide bridges involving Cys18 and either Cys53 or Cys163. We then investigated the roles of the two latter residues, which are conserved in the *Deinococcus* genus [5], to identify which of the two could be the resolving one involved in disulfide isomerization. Very surprisingly, we observed that compared to non-mutated DdMsrA the kinetic parameters of C53S and C163S variants were not affected in the presence of DdTrx1 and only slightly reduced in the presence of DdTrx2 (Table 1), clearly revealing the participation of both Cys in the recycling process by Trx of DdMsrA activity. We can thus hypothesize that following MetO reduction by Cys18, a first bridge is formed with Cys21 and followed by an isomerization step with indifferently Cys53 or Cys163 (i.e., two possible Cys-triad catalytic mechanisms), and subsequent reduction by Trx (Fig. 8A). Moreover, we also show that DdTrx1, and to a lesser extent DdTrx2, are able to form heterodimers with the DdMsrA variant lacking both Cys18 and Cys21 (Fig. 3B-C, lanes 8), indicating that a disulfide bridge, that can be reduced by Trx, is formed in this variant. This prompted us to conclude that DdMsrA recycling could proceed via a second disulfide bridge isomerization step involving the two distal Cys (i.e., a Cys tetrad mechanism), and reduction of this bridge by Trx (Fig. 8B). In line with this conclusion, we observed that following incubation with MetO and migration in non-reducing conditions, non-mutated DdMsrA exhibited a greater electrophoretic mobility than those noticed for single C21S, C53S and C163S variants (Fig. 5). This observation reveals a distinct redox status for DdMsrA that could result from the presence of two disulfide bridges (C18-C21 and C53-C163) following reduction of MetO as supported by the fact that most of the DdMsrA pool is not alkylated when mPEG-maleimide treatment was performed following incubation with MetO (Fig. 6B, lane 3). Taken together, these data support the possibility of a second disulfide isomerization step in the DdMsrA recycling process, in which either Cys53 or Cys163 functions as a third resolving Cys.

Our data unveil MsrA recycling mechanisms implicating four Cys residues that have not been described up to now [32], further highlighting the diversity of the regeneration mechanisms for this type of Msr. Regarding the position of distal resolving Cys in DdMsrA (Cys53 and Cys163) compared to those in *E. coli*, superposition of *E. coli* MsrA X-ray structure [53] and AlphaFold2 DdMsrA model (Fig. 7, Fig. S6D) reveals a similar position for Cys163 and Cys198 in *Deinococcus* and *E. coli* enzymes, respectively. But strikingly, there is no Cys residue in DdMsrA at a position “equivalent” to that of the second resolving one in EcMsrA (Cys206), and the second resolving Cys53 in DdMsrA has the same position as EcMsrA Cys86, which however is not involved in the recycling process [35]. Similarly, the Cys residues that have the same position as DdMsrA resolving Cys53 in *B. subtilis* MsrA (Cys49) and in *L. interrogans* MsrA1 (Cys57) have not been proposed to play a role in MsrA recycling [32,37]. The rate-limiting step in MsrA activity regeneration takes place in the Trx-dependent recycling process [32] and the diversity of Cys distribution and number in MsrA proteins very likely modulates the efficiency of this step [37]. The Cys tetrad-based recycling mechanisms for DdMsrA and the use of two distal resolving Cys could favor MetO binding to the catalytic site due to the absence of steric

<figure><img src="image_17.png" /><figcaption>**Fig. 8.** Proposed recycling mechanisms of DdMsrA by DdTrxs. The first step consists of MetO reduction with the concomitant release of Met and formation of a sulfenic acid intermediate on catalytic Cys18 (1). The sulfenic acid is then attacked by the first resolving Cys21 leading to the release of water and the formation of a disulfide bridge between Cys18 and Cys21 (2). Then, a disulfide bridge isomerization step occurs involving Cys21 with either Cys53 (3) or Cys163 (3'). In the case of recycling by a Cys triad (A), these disulfide bridges are reduced by Trx (4 and 4') leading to oxidation of Trx and regeneration of an active MsrA form (5 and 5'). In the case of recycling by a Cys tetrad (B), a second disulfide isomerization step involving Cys53 and Cys163 occurs (4 and 4'). Reduction of this bridge by Trx (5) leads to oxidation of Trx and regeneration of an active MsrA form (6).</figcaption></figure>

hindrance resulting from a disulfide bridge in the vicinity, and also facilitate the accessibility and/or the reactivity of the disulfide bridge(s) for reduction by Trx. Thus, we might hypothesize that MetO reduction by Cys18 and reaction with Cys21 occur concomitantly to the reduction of the Cys53-Cys163 disulfide bond by Trx. Based on the conservation of the four Cys residues, a similar mechanism very likely applies to all MsrA proteins from deinococcales. In these radiation-tolerant bacteria, this could contribute to highly efficient protein repair and/or redox signaling by Msrs, which are essential actors in responses to environmental constraints in prokaryotes and eukaryotes [20–22,54].

## Funding

This work was funded by a transverse project of the BIAM Institute (PR, LB) that also supported CC's Master 2 internship, by Transverse Division No. 4 (Radiobiology) of the French Alternative Energies and Atomic Energy Commission (Programme No. 4 Radiobiologie) (LB, AdG) and a grant from the Agence Nationale de la Recherche (ANR-19-CE12-0010) (AdG, LB).

## CRediT authorship contribution statement

Pascal Rey: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project
administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Nicolas Rouhier:** Writing – review & editing, Methodology. **Chloé Carassus:** Investigation. **Arjan de Groot:** Writing – review & editing, Writing – original draft, Visualization, Investigation, Funding acquisition. **Laurence Blanchard:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank Océane Reille for production and purification of various protein variants and David Lemaire for mass spectrometry analyses. We are grateful to Drs Christophe Carles and David Pignol for constant support and interest.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbagap.2025.141063.

## Data availability

Data will be made available on request.

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