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

Zhemin Zhou,Yoshiteru Hashimoto,Michihiko Kobayashi

doi:10.1074/jbc.m808464200

Zhemin Zhou, Yoshiteru Hashimoto + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m808464200

Copy DOI

Abstract

The incorporation of cobalt into low molecular mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 has been found to depend on the alpha-subunit exchange between cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues) and its cobalt-containing mediator (holo-NhlAE containing Cys-SO(2)(-) and Cys-SO(-) metal ligands), this novel mode of post-translational maturation having been named self-subunit swapping, and NhlE having been recognized as a self-subunit swapping chaperone (Zhou, Z., Hashimoto, Y., Shiraki, K., and Kobayashi, M. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 14849-14854). We discovered here that cobalt was inserted into both the cobalt-free NhlAE (apo-NhlAE) and the cobalt-free alpha-subunit (apo-alpha-subunit) in an NhlE-dependent manner in the presence of cobalt and dithiothreitol in vitro. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis revealed that the non-oxidized cysteine residues in apo-NhlAE were post-translationally oxidized after cobalt insertion. These findings suggested that NhlE has two activities, i.e. cobalt insertion and cysteine oxidation. NhlE not only functions as a self-subunit swapping chaperone but also a metallochaperone that includes a redox function. Cobalt insertion and cysteine oxidation occurred under both aerobic and anaerobic conditions when Co(3+) was used as a cobalt donor, suggesting that the oxygen atoms in the oxidized cysteines were derived from water molecules but not from dissolved oxygen. Additionally, we isolated apo-NhlAE after the self-subunit swapping event and found that it was recycled for cobalt transfer into L-NHase.

Highlights

Oxidation of the Metal Ligand Cysteine Residues and Insertion of CobaltCys-SO2Ϫ and Cys-SOϪ structures, respectively [3], and the deprotonated Cys-SO2Ϫ and Cys-SOϪ in the holo-␣-subunit form salt bridges with two arginines of the ␤-subunit (which are conserved in all known Co-type and Fe-type NHases) in the holoenzyme (8 –11)
(Institute). 3 The abbreviations used are: NHase, nitrile hydratase; DTT, dithiothreitol; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; R-apo-␣e2, resultant apo-␣e2; KPB, potassium phosphate buffer; CAM, carboxamidomethylated
In this study we discovered that cobalt was directly inserted into a non-oxidized apo-␣-subunit on the addition of NhlE, which yielded a cobalt-containing cysteine-oxidized NhlAE in vitro, suggesting that NhlE acts as a metallochaperone that is crucial for both posttranslational cysteine oxidation and cobalt incorporation into the ␣-subunit of low molecular mass nitrile hydratase (L-NHase) (Fig. 1(ii))

Summary

Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Cys-SO2Ϫ and Cys-SOϪ structures, respectively [3], and the deprotonated Cys-SO2Ϫ and Cys-SOϪ in the holo-␣-subunit form salt bridges with two arginines of the ␤-subunit (which are conserved in all known Co-type and Fe-type NHases) in the holoenzyme (8 –11). Oxidized cysteine residues Cys-SO2H or Cys-SOH are known to play roles in diverse processes, including signal transduction, oxygen metabolism and the oxidative stress response, transcription regulation, and metal coordination in various proteins such as NADH peroxidase [29, 30], peroxiredoxins [31], hydrogenase [32, 33], and so on (8 –12, 26 –28, 34) Among these enzymes, NHase and thiocyanate hydrolase are intriguing ones because they possess both Cys-SO2H and Cys-SOH as ligands of the metal center and neither residue plays any catalytic redox role at all [29]. In this study we discovered that cobalt was directly inserted into a non-oxidized apo-␣-subunit on the addition of NhlE, which yielded a cobalt-containing cysteine-oxidized NhlAE in vitro, suggesting that NhlE acts as a metallochaperone that is crucial for both posttranslational cysteine oxidation and cobalt incorporation into the ␣-subunit of L-NHase (Fig. 1(ii))

EXPERIMENTAL PROCEDURES

RESULTS

Cobalt content

DTT DTT

DISCUSSION