Methionine sulfoxide reductase A is a stereospecific methionine oxidase

Abstract

Methionine sulfoxide reductase A (MsrA) catalyzes the reduction of methionine sulfoxide to methionine and is specific for the S epimer of methionine sulfoxide. The enzyme participates in defense against oxidative stresses by reducing methionine sulfoxide residues in proteins back to methionine. Because oxidation of methionine residues is reversible, this covalent modification could also function as a mechanism for cellular regulation, provided there exists a stereospecific methionine oxidase. We show that MsrA itself is a stereospecific methionine oxidase, producing S-methionine sulfoxide as its product. MsrA catalyzes its own autooxidation as well as oxidation of free methionine and methionine residues in peptides and proteins. When functioning as a reductase, MsrA fully reverses the oxidations which it catalyzes.

Fig. 1.

Met formation and MsrA mass changes. (A) Met production as a function of MetO concentration. Wild-type MsrA (1 μM) was incubated in 50 mM sodium phosphate, 1 mM DTPA, pH 7.5 without reductant at 37 °C for 30 min. (B) Met production as a function of time. The incubation was as in A with MetO held at 50 μM. (C) Mass analysis from the incubations shown in B. Minus 2 Da species, ◊; -4 Da species, Δ; +12 Da species, ∇.

Fig. 2.

Covalent modifications in the C-terminal residues of MsrA. Unmodified and the +12 Da forms were mapped. Oxidation of Met229 to the sulfoxide was confirmed by sequencing, which also confirmed that the two cysteine residues in peptides 1 and 2 were alkylated while they formed a disulfide in peptides 3 and 4.

Fig. 3.

MsrA is stereospecific for the S epimer. (A) Reduction. A mixture of the S and R epimers of MetO was prepared by incubation of 500 μM l-Met with 1 M hydrogen peroxide for 2 h at 37 °C. The Upper tracing shows the chromatogram of the untreated product. The Lower tracing shows that only the S epimer was reduced when 20 μM l-MetO was incubated with MsrA and 10 mM DTT. (B) Oxidation. 20 μM of the sulfenic acid form of MsrA (C107S/C218S/C227S/Δ228–233) was incubated with 20 μM l-Met for 6 h at 37 °C. The product is shown in the Upper tracing, and its complete reduction upon addition of 10 mM DTT in the Lower tracing. For both A and B, incubation with 10 mM DTT alone had no effect.

Fig. 4.

Potential mechanism for regulation of the reductase and oxidase reactions. When thioredoxin has access to the carboxy-domain resolving cysteines, MsrA functions as a reductase. When access of thioredoxin is blocked as a consequence of a conformational change induced by a reversible covalent modification or, as shown, by the binding of a another protein, MsrA functions as an oxidase.

Fig. 5.

Proposed mechanisms for the oxidase and reductase reactions. The reductase mechanism shown on the right is an extension of that proposed by Branlant and coworkers (18). In particular, we propose that the oxygen atom on the sulfenic acid is derived from a water molecule rather than methionine sulfoxide. At the initiation of the reductase reaction (II), the oxygen of the sulfoxide substrate is equidistant (2.6 Å) from the carboxylate oxygen of Glu115 and the hydroxyl of Tyr103 (II) (34). Glu115 acts as a proton donor to the sulfoxide, and the protonated hydroxyl sulfonium intermediate (III) is stabilized by hydrogen bonding with Tyr103. Nucleophilic attack of the hydroxyl sulfonium intermediate by the thiolate of Cys72 forms the sulfurane intermediate (IV) (–44). It has been suggested that the sulfurane hydroxyl group could be protonated by a second methanthiol molecule and form the sulfonium intermediate upon loss of water. The pK_a of methanthiol and the hydroxyl of tyrosine are both about 10.2, leading us to suggest that Tyr103 is the second proton donor, facilitating formation of the sulfonium intermediate upon leaving of a water molecule which would be exchangeable with solvent water (V). Another water molecule, seen in several crystal structures of MsrA is sited 2.8 Å from Cys72 on the opposite side from the bound substrate. This location places it in perfect position to react with the sulfonium intermediate to form the sulfenic acid on Cys72 and release Met as a product (VI). After the release of Met, the Cys72 sulfenic acid form (VII) can be reduced back to the thiolate (I) by reducing agents, including the C-terminal resolving cysteines or DTT. The sulfenic acid can also undergo reversible dehydration to form a sulfenamide by reaction with either a side chain amino group or an amide group in the backbone, a reaction favored in the electrospray mass spectrometer. The left side of the figure shows the proposed mechanism for the MsrA oxidase. Met binds to the sulfenic acid form of MsrA in the same orientation as MetO does in the reductase cycle and with water bound to Glu115 and Tyr103 (VIII). A lone pair of electrons on the Met sulfur attacks the sulfenic sulfur in a SN2 (bimolecular nucleophilic substitution) manner to form the positively charged sulfonium intermediate (IX) and release a molecule of water. Another molecule of water, presumably again bound in the pocket with Glu115 and Tyr103, attacks the sulfonium sulfur to form the sulfurane intermediate (X). Glu115 then acts as a general base to abstract a proton from the hydroxyl group of the sulfurane intermediate. The sulfur–sulfur bond breaks to form MetO and Cys72 is regenerated (XI). With release of MetO, the reduced form of MsrA is formed (I).