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.
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.
Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Zhemin
Z
Zhou
Yoshiteru
Y
Hashimoto
Michihiko
M
Kobayashi
D000332:Aerobiosis; D000693:Anaerobiosis; D001051:Apoenzymes / Q000737:chemistry / Q000302:isolation & purification; D003035:Cobalt / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation; D006836:Hydro-Lyases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry*; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism*; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001051:Apoenzymes; D008024:Ligands; D008667:Metalloproteins; D018832:Molecular Chaperones; D021122:Protein Subunits; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase; D003545:Cysteine