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 symmetrical dimethylation of residue Arg-85 of the 49-kDa subunit of bovine complex I.A and B, spectra of fragments produced, respectively, by ETD of a quadruply charged ion (447.99 m/z) and by HCD of a triply charged ion (596.99 m/z), both derived from the tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit. In A, a singly charged ion (447.12 m/z) is a contaminant of the precursor (M+4H)4+ ion. In B, ● denotes ions arising by internal fragmentation. The ions z4-z5, c10-c11, and y4-y5 demonstrate dimethylation of residue Arg-85 of the 49-kDa subunit. The losses of the neutral fragments with masses of 31 (monomethylamine) from y5-y10 and 70 (dimethylcarbodiimide) from y9 and y10, labeled * and ∧ respectively, define the modification as the symmetrical dimethylation of the guanidino group of residue Arg-85. In the insets, the fragment ions are mapped onto the amino acid sequence; c is carbamidomethylcysteine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Characterization of the symmetrical dimethylation of residue Arg-85 of the 49-kDa subunit of bovine complex I.A and B, spectra of fragments produced, respectively, by ETD of a quadruply charged ion (447.99 m/z) and by HCD of a triply charged ion (596.99 m/z), both derived from the tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit. In A, a singly charged ion (447.12 m/z) is a contaminant of the precursor (M+4H)4+ ion. In B, ● denotes ions arising by internal fragmentation. The ions z4-z5, c10-c11, and y4-y5 demonstrate dimethylation of residue Arg-85 of the 49-kDa subunit. The losses of the neutral fragments with masses of 31 (monomethylamine) from y5-y10 and 70 (dimethylcarbodiimide) from y9 and y10, labeled * and ∧ respectively, define the modification as the symmetrical dimethylation of the guanidino group of residue Arg-85. In the insets, the fragment ions are mapped onto the amino acid sequence; c is carbamidomethylcysteine.

Mentions: In the tryptic digest of the 49-kDa subunit, an unexplained peptide with m/z value of 1788.95 was discovered by MALDI-TOF analysis. Its mass corresponds to the calculated mass of the tryptic peptide from residues 75–89 plus 28 Da. Fragmentation of this peptide (Fig. 3) confirmed that its sequence, KCDPHIGLLHRGTEK, corresponds to residues 75–89 of the 49-kDa protein with the additional mass of 28 Da associated with residue Arg-85, suggesting that its guanidino group is dimethylated. The fragment ion spectra of the peptide (Fig. 3) contained several y-ions resulting from the neutral loss of 31 and 70 mass units, corresponding to the loss of methylamine and dimethylcarbodiimide, respectively, which are diagnostic of two methyl groups attached symmetrically to the ω-NG and ω-NG′ nitrogen atoms of the guanidino group of an arginine residue (32, 33). Thus, it was concluded that residue Arg-85 of the 49-kDa subunit of bovine complex I is symmetrically dimethylated. There was no evidence in the mass spectrum of y-ions arising from the loss of 45 Da corresponding to dimethylamine, which would be diagnostic of an arginine residue asymmetrically dimethylated on one of the Nω nitrogen atoms (32, 33). Also, none of the spectra contained evidence for the unmethylated or monomethylated forms of residue Arg-85, and so the residue appears to be completely symmetrically dimethylated. The equivalent arginine residues in the human, P. pastoris, P. denitrificans, and E. coli enzymes are conserved (Fig. 4). The symmetrical dimethylation of the arginine residue is also conserved in the human, P. pastoris, and P. denitrificans complexes I (Fig. 5), but the equivalent residue in the NuoCD subunit of the E. coli enzyme is not modified (Fig. 5).


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 symmetrical dimethylation of residue Arg-85 of the 49-kDa subunit of bovine complex I.A and B, spectra of fragments produced, respectively, by ETD of a quadruply charged ion (447.99 m/z) and by HCD of a triply charged ion (596.99 m/z), both derived from the tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit. In A, a singly charged ion (447.12 m/z) is a contaminant of the precursor (M+4H)4+ ion. In B, ● denotes ions arising by internal fragmentation. The ions z4-z5, c10-c11, and y4-y5 demonstrate dimethylation of residue Arg-85 of the 49-kDa subunit. The losses of the neutral fragments with masses of 31 (monomethylamine) from y5-y10 and 70 (dimethylcarbodiimide) from y9 and y10, labeled * and ∧ respectively, define the modification as the symmetrical dimethylation of the guanidino group of residue Arg-85. In the insets, the fragment ions are mapped onto the amino acid sequence; c is carbamidomethylcysteine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Characterization of the symmetrical dimethylation of residue Arg-85 of the 49-kDa subunit of bovine complex I.A and B, spectra of fragments produced, respectively, by ETD of a quadruply charged ion (447.99 m/z) and by HCD of a triply charged ion (596.99 m/z), both derived from the tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit. In A, a singly charged ion (447.12 m/z) is a contaminant of the precursor (M+4H)4+ ion. In B, ● denotes ions arising by internal fragmentation. The ions z4-z5, c10-c11, and y4-y5 demonstrate dimethylation of residue Arg-85 of the 49-kDa subunit. The losses of the neutral fragments with masses of 31 (monomethylamine) from y5-y10 and 70 (dimethylcarbodiimide) from y9 and y10, labeled * and ∧ respectively, define the modification as the symmetrical dimethylation of the guanidino group of residue Arg-85. In the insets, the fragment ions are mapped onto the amino acid sequence; c is carbamidomethylcysteine.
Mentions: In the tryptic digest of the 49-kDa subunit, an unexplained peptide with m/z value of 1788.95 was discovered by MALDI-TOF analysis. Its mass corresponds to the calculated mass of the tryptic peptide from residues 75–89 plus 28 Da. Fragmentation of this peptide (Fig. 3) confirmed that its sequence, KCDPHIGLLHRGTEK, corresponds to residues 75–89 of the 49-kDa protein with the additional mass of 28 Da associated with residue Arg-85, suggesting that its guanidino group is dimethylated. The fragment ion spectra of the peptide (Fig. 3) contained several y-ions resulting from the neutral loss of 31 and 70 mass units, corresponding to the loss of methylamine and dimethylcarbodiimide, respectively, which are diagnostic of two methyl groups attached symmetrically to the ω-NG and ω-NG′ nitrogen atoms of the guanidino group of an arginine residue (32, 33). Thus, it was concluded that residue Arg-85 of the 49-kDa subunit of bovine complex I is symmetrically dimethylated. There was no evidence in the mass spectrum of y-ions arising from the loss of 45 Da corresponding to dimethylamine, which would be diagnostic of an arginine residue asymmetrically dimethylated on one of the Nω nitrogen atoms (32, 33). Also, none of the spectra contained evidence for the unmethylated or monomethylated forms of residue Arg-85, and so the residue appears to be completely symmetrically dimethylated. The equivalent arginine residues in the human, P. pastoris, P. denitrificans, and E. coli enzymes are conserved (Fig. 4). The symmetrical dimethylation of the arginine residue is also conserved in the human, P. pastoris, and P. denitrificans complexes I (Fig. 5), but the equivalent residue in the NuoCD subunit of the E. coli enzyme is not modified (Fig. 5).

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