Post‐translational modification is essential for catalytic activity of nitrile hydratase

Abstract

Nitrile hydratase from Rhodococcus sp. N-771 is an ab heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, aCys112 and aCys114 are modified to a cysteine sulfinic acid~Cys-SO 2 H! and a cysteine sulfenic acid~Cys-SOH!, respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The ab complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI-LC0MS analysis showed that the anaerobically reconstituted ab complex did not have the modification of aCys112-SO 2 H and aerobic incubation induced the modification. The activity of the reconstituted ab complex correlated with the amount of aCys112-SO 2 H. Furthermore, ESI-LC0MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that aCys114 is modified to Cys-SOH together with the sulfinic acid modification of aCys112. These results suggest that aCys112 and aCys114 are spontaneously oxidized to Cys-SO 2 H and Cys-SOH, respectively, and aCys112-SO 2 H is responsible for the catalytic activity solely or in combination with aCys114-SOH.

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

“…We discovered that NhlE facilitates Cys-SOH and Cys-SO 2 H oxidation post-translationally and for the first time succeeded in making cysteine residues in a protein undergo oxidation to stable Cys-SOH and Cys-SO 2 H in a protein-dependent manner in vitro, whereas enhancement of the spontaneous oxidation of cysteine residues in the ␣-subunit of Fe-NHase in Rhodococcus sp. N-771 in the presence of ferric citrate in vitro has been observed (34). Cobalt exists as a non-corrin low-spin Co 3ϩ ion in H-NHase in R. rhodochrous J1 (40) and NHase in P. putida NRRL-18668 (7), suggesting that the holo-␣-subunit of L-NHase (holo-␣ 2 ␤ 2 ) and the holo-␣-subunit of holo-␣e 2 (which is the source of that of holo-␣ 2 ␤ 2 ) should also have a non-corrin low-spin Co 3ϩ ion.…”

“…5), suggesting that Cys-112 in apo-␣e 2 was oxidized to Cys-112-SO 2 Ϫ in R-apo-␣e 2 . Although the occurrence of Cys-114-SOH oxidation has not been confirmed because of the chemical instability (34), this finding strongly suggests that oxidized cysteine residues (␣Cys-112-SO 2 Ϫ and ␣Cys-114-SO Ϫ ) exist in R-apo-␣e 2 . Necessity of NhlE for Cobalt Incorporation into the ␣-SubunitTo determine whether cobalt could be directly inserted into the apo-␣-subunit in the absence of NhlE or not, we separated the ␣-subunit and NhlE through denaturation and renaturation (20).…”

“…Among these enzymes, NHase and thiocyanate hydrolase are intriguing ones because they possess both Cys-SO 2 H and Cys-SOH as ligands of the metal center and neither residue plays any catalytic redox role at all (29). Although the oxidized cysteine residues in both enzymes are essential for their activities (20,34), the mechanism(s) involved in the post-translational oxidation of both residues remains unclear.…”

The Redox Biochemistry of Protein Sulfenylation and Sulfinylation

“…Structural analysis reveals that NHase contains an Fe III or Co III active site, in which three Cys residues, with three different oxidation states (RSH, RSOH, and RSO 2 H), contribute to the coordination of the metal ion (74,75). The fully reduced enzyme appears inactive, suggesting that Cys sulfenylation and sulfinylation are critical in maintaining the catalytic activity of NHase (76), probably by increasing the Lewis acidity of the metal ion. An analogous motif was more recently found in the catalytic site of thiocyanate hydrolase (SCNase), which incorporates Co III only after Cys oxidation (77).…”

Fragmentation of protonated ions of peptides containing cysteine, cysteine sulfinic acid, and cysteine sulfonic acid

“…The Cys-SOH is unstable and it reacts with any accessible thiol to form a disulfide or undergoes further oxidation to give cysteine sulfinic acid (Cys-SO 2 H) and cysteine sulfonic acid (Cys-SO 3 H) [2]. The formation of Cys-SO 2 H and Cys-SO 3 H has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) [8] and nitrile hydratase [9]. The oxidation to Cys-SO 2 H and Cys-SO 3 H is generally thought to be irreversible [2].…”