Limits...
Post-translational modifications near the quinone binding site of mammalian complex I.

Carroll J, Ding S, Fearnley IM, Walker JE - J. Biol. Chem. (2013)

Bottom Line: An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties.Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone.Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.

View Article: PubMed Central - PubMed

Affiliation: Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.

Show MeSH

Related in: MedlinePlus

Characterization of the hydroxylation of residue Arg-73 of the PSST subunit of human complex I and the unmodified residue in the E. coli NuoB subunit.A, ETD fragmentation spectrum of a triply charged ion, m/z 546.62, generated by cleavage of the human PSST protein with Asp-N. The ions z6-z7 and c7-c8 show that the +16 Da modification is associated with residue Arg-73. B, spectrum of fragments produced by ETD from a triply charged ion (m/z 615.00) from a peptide corresponding to residues 77–93 of the E. coli NuoB subunit. The series of fragment ions identifies the peptide and excludes modification of residue Arg-87. In the insets, the fragment ions are mapped onto the amino acid sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750175&req=5

Figure 8: Characterization of the hydroxylation of residue Arg-73 of the PSST subunit of human complex I and the unmodified residue in the E. coli NuoB subunit.A, ETD fragmentation spectrum of a triply charged ion, m/z 546.62, generated by cleavage of the human PSST protein with Asp-N. The ions z6-z7 and c7-c8 show that the +16 Da modification is associated with residue Arg-73. B, spectrum of fragments produced by ETD from a triply charged ion (m/z 615.00) from a peptide corresponding to residues 77–93 of the E. coli NuoB subunit. The series of fragment ions identifies the peptide and excludes modification of residue Arg-87. In the insets, the fragment ions are mapped onto the amino acid sequence.

Mentions: The digest of the bovine PSST subunit with protease Asp-N contained a hitherto unassigned peptide with m/z 1637.84. This value corresponds to a singly charged ion arising from residues 70–83 of the PSST subunit, with an additional mass of 16 Da. The spectrum of the fragments produced from the triply charged ion (Fig. 6) confirmed that the sequence of the peptide was DRFGVVFRASPRQS with an additional 16 mass units, corresponding to a hydroxyl group associated with residue Arg-77. Thus, this residue is probably hydroxyarginine. There was no evidence in any of the spectra for the unmodified form, and so it appears that this modification is quantitative. Residue Arg-77 is conserved in the human, P. pastoris, and E. coli enzymes (Fig. 7). An additional mass of 16 Da is associated with this residue in the human protein, and so Arg-73 also appears to be hydroxylated in the human PSST subunit (Fig. 8). The mass spectral data do not provide evidence about which atom in the side chain of bovine Arg-77 or human Arg-73 is hydroxylated. In the E. coli enzyme, the experimental and calculated molecular masses of the NuoB subunit are in agreement, and the equivalent arginine residue Arg-87 is unmodified (Fig. 8), and in the P. pastoris enzyme, the modification is not present either (26). It is not known whether the Nqo6 subunit of complex I from P. denitrificans is modified.


Post-translational modifications near the quinone binding site of mammalian complex I.

Carroll J, Ding S, Fearnley IM, Walker JE - J. Biol. Chem. (2013)

Characterization of the hydroxylation of residue Arg-73 of the PSST subunit of human complex I and the unmodified residue in the E. coli NuoB subunit.A, ETD fragmentation spectrum of a triply charged ion, m/z 546.62, generated by cleavage of the human PSST protein with Asp-N. The ions z6-z7 and c7-c8 show that the +16 Da modification is associated with residue Arg-73. B, spectrum of fragments produced by ETD from a triply charged ion (m/z 615.00) from a peptide corresponding to residues 77–93 of the E. coli NuoB subunit. The series of fragment ions identifies the peptide and excludes modification of residue Arg-87. In the insets, the fragment ions are mapped onto the amino acid sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750175&req=5

Figure 8: Characterization of the hydroxylation of residue Arg-73 of the PSST subunit of human complex I and the unmodified residue in the E. coli NuoB subunit.A, ETD fragmentation spectrum of a triply charged ion, m/z 546.62, generated by cleavage of the human PSST protein with Asp-N. The ions z6-z7 and c7-c8 show that the +16 Da modification is associated with residue Arg-73. B, spectrum of fragments produced by ETD from a triply charged ion (m/z 615.00) from a peptide corresponding to residues 77–93 of the E. coli NuoB subunit. The series of fragment ions identifies the peptide and excludes modification of residue Arg-87. In the insets, the fragment ions are mapped onto the amino acid sequence.
Mentions: The digest of the bovine PSST subunit with protease Asp-N contained a hitherto unassigned peptide with m/z 1637.84. This value corresponds to a singly charged ion arising from residues 70–83 of the PSST subunit, with an additional mass of 16 Da. The spectrum of the fragments produced from the triply charged ion (Fig. 6) confirmed that the sequence of the peptide was DRFGVVFRASPRQS with an additional 16 mass units, corresponding to a hydroxyl group associated with residue Arg-77. Thus, this residue is probably hydroxyarginine. There was no evidence in any of the spectra for the unmodified form, and so it appears that this modification is quantitative. Residue Arg-77 is conserved in the human, P. pastoris, and E. coli enzymes (Fig. 7). An additional mass of 16 Da is associated with this residue in the human protein, and so Arg-73 also appears to be hydroxylated in the human PSST subunit (Fig. 8). The mass spectral data do not provide evidence about which atom in the side chain of bovine Arg-77 or human Arg-73 is hydroxylated. In the E. coli enzyme, the experimental and calculated molecular masses of the NuoB subunit are in agreement, and the equivalent arginine residue Arg-87 is unmodified (Fig. 8), and in the P. pastoris enzyme, the modification is not present either (26). It is not known whether the Nqo6 subunit of complex I from P. denitrificans is modified.

Bottom Line: An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties.Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone.Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.

View Article: PubMed Central - PubMed

Affiliation: Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.

Show MeSH
Related in: MedlinePlus