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

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.