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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.

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Reinvestigation of the sequences of the 49-kDa and PSST subunits of bovine complex I. The sequences of peptides in proteolytic digests of the proteins were analyzed by tandem mass spectrometry. A, the 49-kDa subunit; B, the PSST subunit. Segments highlighted in green, yellow, and red correspond to regions where the sequence was confirmed with high, medium, or low confidence, respectively. No evidence was found in these experiments for the regions that are not highlighted. In subsequent analyses presented below, a tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit (underlined) was found to be post-translationally modified by symmetrical dimethylation of the guanidino group of residue Arg-85, and an Asp-N peptide from the PSST subunit corresponding to residues 70–83 (underlined) was shown to bear a hydroxyl group on residue Arg-77. The modified residues are highlighted in light blue. In the 49-kDa subunit, both arginine and glutamine have been found at residue 129.
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Figure 1: Reinvestigation of the sequences of the 49-kDa and PSST subunits of bovine complex I. The sequences of peptides in proteolytic digests of the proteins were analyzed by tandem mass spectrometry. A, the 49-kDa subunit; B, the PSST subunit. Segments highlighted in green, yellow, and red correspond to regions where the sequence was confirmed with high, medium, or low confidence, respectively. No evidence was found in these experiments for the regions that are not highlighted. In subsequent analyses presented below, a tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit (underlined) was found to be post-translationally modified by symmetrical dimethylation of the guanidino group of residue Arg-85, and an Asp-N peptide from the PSST subunit corresponding to residues 70–83 (underlined) was shown to bear a hydroxyl group on residue Arg-77. The modified residues are highlighted in light blue. In the 49-kDa subunit, both arginine and glutamine have been found at residue 129.

Mentions: The mass spectrometric analysis of enzymic digests of both the 49-kDa and the PSST subunits with trypsin, chymotrypsin, and protease Asp-N validated almost the entire amino acid sequences of both proteins with high or medium confidence (Fig. 1). However, residues 76–94, 295–317, and 330–339 in the 49-kDa subunit and 68–81 and 170–179 in the PSST subunit were either not covered at all (residues 84–90 in the 49-kDa subunit and 170–172 and 179 in the PSST subunit), or they were covered sparsely. Therefore, these regions became the focus of attention in the search for an explanation of the discrepancies between experimentally measured and calculated intact protein masses. An additional complexity in the case of the bovine 49-kDa subunit is that there are two isoforms differing by the single amino acid substitution R129Q, presumably arising from different alleles in the bovine population (31). Tryptic peptides corresponding to both isoforms were detected (Fig. 2), but the predominant component, as estimated from the areas of the ion peaks, was the isoform with Arg-129. The calculated masses of the isoforms of the 49-kDa subunit with Arg-129 and Gln-129 are 28 and 56 Da, respectively, less than the measured intact molecular mass (2, 23).


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

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

Reinvestigation of the sequences of the 49-kDa and PSST subunits of bovine complex I. The sequences of peptides in proteolytic digests of the proteins were analyzed by tandem mass spectrometry. A, the 49-kDa subunit; B, the PSST subunit. Segments highlighted in green, yellow, and red correspond to regions where the sequence was confirmed with high, medium, or low confidence, respectively. No evidence was found in these experiments for the regions that are not highlighted. In subsequent analyses presented below, a tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit (underlined) was found to be post-translationally modified by symmetrical dimethylation of the guanidino group of residue Arg-85, and an Asp-N peptide from the PSST subunit corresponding to residues 70–83 (underlined) was shown to bear a hydroxyl group on residue Arg-77. The modified residues are highlighted in light blue. In the 49-kDa subunit, both arginine and glutamine have been found at residue 129.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Reinvestigation of the sequences of the 49-kDa and PSST subunits of bovine complex I. The sequences of peptides in proteolytic digests of the proteins were analyzed by tandem mass spectrometry. A, the 49-kDa subunit; B, the PSST subunit. Segments highlighted in green, yellow, and red correspond to regions where the sequence was confirmed with high, medium, or low confidence, respectively. No evidence was found in these experiments for the regions that are not highlighted. In subsequent analyses presented below, a tryptic peptide corresponding to residues 75–89 of the 49-kDa subunit (underlined) was found to be post-translationally modified by symmetrical dimethylation of the guanidino group of residue Arg-85, and an Asp-N peptide from the PSST subunit corresponding to residues 70–83 (underlined) was shown to bear a hydroxyl group on residue Arg-77. The modified residues are highlighted in light blue. In the 49-kDa subunit, both arginine and glutamine have been found at residue 129.
Mentions: The mass spectrometric analysis of enzymic digests of both the 49-kDa and the PSST subunits with trypsin, chymotrypsin, and protease Asp-N validated almost the entire amino acid sequences of both proteins with high or medium confidence (Fig. 1). However, residues 76–94, 295–317, and 330–339 in the 49-kDa subunit and 68–81 and 170–179 in the PSST subunit were either not covered at all (residues 84–90 in the 49-kDa subunit and 170–172 and 179 in the PSST subunit), or they were covered sparsely. Therefore, these regions became the focus of attention in the search for an explanation of the discrepancies between experimentally measured and calculated intact protein masses. An additional complexity in the case of the bovine 49-kDa subunit is that there are two isoforms differing by the single amino acid substitution R129Q, presumably arising from different alleles in the bovine population (31). Tryptic peptides corresponding to both isoforms were detected (Fig. 2), but the predominant component, as estimated from the areas of the ion peaks, was the isoform with Arg-129. The calculated masses of the isoforms of the 49-kDa subunit with Arg-129 and Gln-129 are 28 and 56 Da, respectively, less than the measured intact molecular mass (2, 23).

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