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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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Evidence for the presence of two isoforms of the 49-kDa subunit in preparations of bovine complex I. The isoforms differ by the presence of arginine or glutamine at position 129 of the mature protein. A and B, fragment ion spectra produced by CID of doubly charged ions with m/z 524.32 and 538.34 arising from the tryptic peptides corresponding to residues 125–133 of the 49-kDa subunit. The fragment ions y4-y5 and b4-b5 define residue 129 as Gln in A and as Arg in B, in the tryptic peptide sequence LLNI(Q/R)PPPR. In B, the y-ions appear as the y-ion series and as a y-17 series arising from the loss of ammonia. In the insets, the identified b- and y- fragment ions are mapped onto the amino acid sequence.
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Figure 2: Evidence for the presence of two isoforms of the 49-kDa subunit in preparations of bovine complex I. The isoforms differ by the presence of arginine or glutamine at position 129 of the mature protein. A and B, fragment ion spectra produced by CID of doubly charged ions with m/z 524.32 and 538.34 arising from the tryptic peptides corresponding to residues 125–133 of the 49-kDa subunit. The fragment ions y4-y5 and b4-b5 define residue 129 as Gln in A and as Arg in B, in the tryptic peptide sequence LLNI(Q/R)PPPR. In B, the y-ions appear as the y-ion series and as a y-17 series arising from the loss of ammonia. In the insets, the identified b- and y- fragment ions are mapped onto the amino acid sequence.

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)

Evidence for the presence of two isoforms of the 49-kDa subunit in preparations of bovine complex I. The isoforms differ by the presence of arginine or glutamine at position 129 of the mature protein. A and B, fragment ion spectra produced by CID of doubly charged ions with m/z 524.32 and 538.34 arising from the tryptic peptides corresponding to residues 125–133 of the 49-kDa subunit. The fragment ions y4-y5 and b4-b5 define residue 129 as Gln in A and as Arg in B, in the tryptic peptide sequence LLNI(Q/R)PPPR. In B, the y-ions appear as the y-ion series and as a y-17 series arising from the loss of ammonia. In the insets, the identified b- and y- 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 2: Evidence for the presence of two isoforms of the 49-kDa subunit in preparations of bovine complex I. The isoforms differ by the presence of arginine or glutamine at position 129 of the mature protein. A and B, fragment ion spectra produced by CID of doubly charged ions with m/z 524.32 and 538.34 arising from the tryptic peptides corresponding to residues 125–133 of the 49-kDa subunit. The fragment ions y4-y5 and b4-b5 define residue 129 as Gln in A and as Arg in B, in the tryptic peptide sequence LLNI(Q/R)PPPR. In B, the y-ions appear as the y-ion series and as a y-17 series arising from the loss of ammonia. In the insets, the identified b- and y- fragment ions are mapped onto the amino acid sequence.
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