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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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Locations of the modified arginine residues in the vicinity of the quinone binding site of bovine complex I.A, the outline of the structure of complex I illustrating the pathway of electron transfer from NADH to coenzyme Q via flavin mononucleotide and a chain of seven Fe-S clusters (open circles). The terminal Fe-S cluster N2 is labeled. As indicated, the transfer of two electrons one at a time via this pathway is coupled to the translocation of four protons into the mitochondrial intermembrane space from the mitochondrial matrix. The large arrow on the right indicates the direction of view of the quinone binding site in B. B, a detailed view of the quinone binding site involving the 49-kDa, PSST, and ND1 subunits (orange, blue, and green, respectively). The green ND1 subunit is divided into two unequal areas by the intervening quinone binding site, one small and approximately triangular, the other larger and approximately pentangular. The area shaded lighter green indicates that the two dark green areas are joined in front and behind the binding site. B is based upon the structures and positional relationships of the orthologous core proteins, Nqo4, Nqo6, and Nqo8, in the structure of complex I from T. thermophilus (5). The view is along the axis of the membrane arm of complex I away from its junction with the orthologous extrinsic arm (vertical pointing upward into the matrix of the mitochondrion). The approximate position of the phospholipid bilayer of the inner mitochondrial membrane is indicated. The quinone is shown with its head group and part of its side chain bound in a tunnel between the three subunits. The entrance to the tunnel is formed by the transmembrane α-helices 1 and 6 and amphipathic α-helix 1 of subunit Nqo8. The hydroxylated Arg-77 in the bovine PSST subunit is in a loop (not modeled in the structure of the bacterial complex, but linking α-helices 2 and 3). The dimethylated Arg-85 in the bovine 49-kDa subunit is in a loop between β-strand 3 and α-helix 1 close to Fe-S cluster N2 (brown cube), which is attached to the PSST subunit. The arrow indicates the direction of electron flow from the penultimate Fe-S cluster to the terminal cluster, N2.
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Figure 9: Locations of the modified arginine residues in the vicinity of the quinone binding site of bovine complex I.A, the outline of the structure of complex I illustrating the pathway of electron transfer from NADH to coenzyme Q via flavin mononucleotide and a chain of seven Fe-S clusters (open circles). The terminal Fe-S cluster N2 is labeled. As indicated, the transfer of two electrons one at a time via this pathway is coupled to the translocation of four protons into the mitochondrial intermembrane space from the mitochondrial matrix. The large arrow on the right indicates the direction of view of the quinone binding site in B. B, a detailed view of the quinone binding site involving the 49-kDa, PSST, and ND1 subunits (orange, blue, and green, respectively). The green ND1 subunit is divided into two unequal areas by the intervening quinone binding site, one small and approximately triangular, the other larger and approximately pentangular. The area shaded lighter green indicates that the two dark green areas are joined in front and behind the binding site. B is based upon the structures and positional relationships of the orthologous core proteins, Nqo4, Nqo6, and Nqo8, in the structure of complex I from T. thermophilus (5). The view is along the axis of the membrane arm of complex I away from its junction with the orthologous extrinsic arm (vertical pointing upward into the matrix of the mitochondrion). The approximate position of the phospholipid bilayer of the inner mitochondrial membrane is indicated. The quinone is shown with its head group and part of its side chain bound in a tunnel between the three subunits. The entrance to the tunnel is formed by the transmembrane α-helices 1 and 6 and amphipathic α-helix 1 of subunit Nqo8. The hydroxylated Arg-77 in the bovine PSST subunit is in a loop (not modeled in the structure of the bacterial complex, but linking α-helices 2 and 3). The dimethylated Arg-85 in the bovine 49-kDa subunit is in a loop between β-strand 3 and α-helix 1 close to Fe-S cluster N2 (brown cube), which is attached to the PSST subunit. The arrow indicates the direction of electron flow from the penultimate Fe-S cluster to the terminal cluster, N2.

Mentions: Currently, there is no high resolution structural information about mammalian complex I, but the structure of complex I from the thermophilic bacterium Thermus thermophilus has been described (5), and it contains the structures of the orthologs of all of the core subunits of the mammalian complex, including those of Nqo4 and Nqo6, the orthologs of the 49-kDa and PSST subunit, respectively. The sequences of Nqo4 and the 49-kDa subunit and Nqo6 and the PSST subunit are 61 and 64% conserved, respectively, with 42 and 48% identical, respectively, (supplemental Fig. S1), and therefore, the structure of complex I from T. thermophilus provides a reasonable model for examining the general environments of the modified arginine residues in the 49-kDa and PSST subunits of the bovine and human enzymes. A schematic representation of the region where the modified residues are found is shown in Fig. 9. The dimethylated Arg-85 in the bovine 49-kDa subunit replaces a threonine residue in the bacterial enzyme in a loop between β-strand 3 and α-helix 1, close to (∼7 Å) Fe-S cluster N2, which is attached to the PSST subunit; the larger side chain of the methylated arginine could be even closer. The methylation of an arginine residue increases the hydrophobicity and solvent-accessible surface of the side chain, reduces its potential to form hydrogen bonds, and lowers its pI value slightly (53). Cluster N2 is the terminal Fe-S cluster in the chain of seven Fe-S clusters, and if for example the methylated arginine residue were close to one of the side chains of the cysteine residues that ligand the cluster, the dimethylated arginine residue might conceivably influence its redox potential. Another possibility is that the methylated residue influences the assembly of complex I, and the association of human pathogenic mutations in putative protein methylases that lead to dysfunction of complex I (48) makes this an attractive possibility. The hydroxylated Arg-77 in the bovine PSST subunit is in a loop (not resolved in the structure of the bacterial complex) linking α-helices 2 and 3 close to the tunnel, also involving the ND1 and 49-kDa subunits, in which oxidized coenzyme Q is thought to bind so as to accept electrons from cluster N2. In the absence of more detailed structural information, it is currently not possible to understand the role of the hydroxylated arginine residue. It is unlikely that the hydroxylation of the arginine residue arises in the tunnel, for example by reaction with reactive oxygen species, as such species are not thought to be generated from the quinone (54), although this conclusion is disputed (55). The restricted access to both modified residues would make enzymatic modification via the tunnel unlikely, and therefore, it is much more probable that the modifications occur in the cytoplasm at any one of the stages following synthesis, during or following import into the mitochondrion or during assembly into complex I. The identification of the modifying enzymes is likely to help in determining the cellular site of modification, and it may help also in understanding the roles of the modified arginine residues.


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

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

Locations of the modified arginine residues in the vicinity of the quinone binding site of bovine complex I.A, the outline of the structure of complex I illustrating the pathway of electron transfer from NADH to coenzyme Q via flavin mononucleotide and a chain of seven Fe-S clusters (open circles). The terminal Fe-S cluster N2 is labeled. As indicated, the transfer of two electrons one at a time via this pathway is coupled to the translocation of four protons into the mitochondrial intermembrane space from the mitochondrial matrix. The large arrow on the right indicates the direction of view of the quinone binding site in B. B, a detailed view of the quinone binding site involving the 49-kDa, PSST, and ND1 subunits (orange, blue, and green, respectively). The green ND1 subunit is divided into two unequal areas by the intervening quinone binding site, one small and approximately triangular, the other larger and approximately pentangular. The area shaded lighter green indicates that the two dark green areas are joined in front and behind the binding site. B is based upon the structures and positional relationships of the orthologous core proteins, Nqo4, Nqo6, and Nqo8, in the structure of complex I from T. thermophilus (5). The view is along the axis of the membrane arm of complex I away from its junction with the orthologous extrinsic arm (vertical pointing upward into the matrix of the mitochondrion). The approximate position of the phospholipid bilayer of the inner mitochondrial membrane is indicated. The quinone is shown with its head group and part of its side chain bound in a tunnel between the three subunits. The entrance to the tunnel is formed by the transmembrane α-helices 1 and 6 and amphipathic α-helix 1 of subunit Nqo8. The hydroxylated Arg-77 in the bovine PSST subunit is in a loop (not modeled in the structure of the bacterial complex, but linking α-helices 2 and 3). The dimethylated Arg-85 in the bovine 49-kDa subunit is in a loop between β-strand 3 and α-helix 1 close to Fe-S cluster N2 (brown cube), which is attached to the PSST subunit. The arrow indicates the direction of electron flow from the penultimate Fe-S cluster to the terminal cluster, N2.
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Figure 9: Locations of the modified arginine residues in the vicinity of the quinone binding site of bovine complex I.A, the outline of the structure of complex I illustrating the pathway of electron transfer from NADH to coenzyme Q via flavin mononucleotide and a chain of seven Fe-S clusters (open circles). The terminal Fe-S cluster N2 is labeled. As indicated, the transfer of two electrons one at a time via this pathway is coupled to the translocation of four protons into the mitochondrial intermembrane space from the mitochondrial matrix. The large arrow on the right indicates the direction of view of the quinone binding site in B. B, a detailed view of the quinone binding site involving the 49-kDa, PSST, and ND1 subunits (orange, blue, and green, respectively). The green ND1 subunit is divided into two unequal areas by the intervening quinone binding site, one small and approximately triangular, the other larger and approximately pentangular. The area shaded lighter green indicates that the two dark green areas are joined in front and behind the binding site. B is based upon the structures and positional relationships of the orthologous core proteins, Nqo4, Nqo6, and Nqo8, in the structure of complex I from T. thermophilus (5). The view is along the axis of the membrane arm of complex I away from its junction with the orthologous extrinsic arm (vertical pointing upward into the matrix of the mitochondrion). The approximate position of the phospholipid bilayer of the inner mitochondrial membrane is indicated. The quinone is shown with its head group and part of its side chain bound in a tunnel between the three subunits. The entrance to the tunnel is formed by the transmembrane α-helices 1 and 6 and amphipathic α-helix 1 of subunit Nqo8. The hydroxylated Arg-77 in the bovine PSST subunit is in a loop (not modeled in the structure of the bacterial complex, but linking α-helices 2 and 3). The dimethylated Arg-85 in the bovine 49-kDa subunit is in a loop between β-strand 3 and α-helix 1 close to Fe-S cluster N2 (brown cube), which is attached to the PSST subunit. The arrow indicates the direction of electron flow from the penultimate Fe-S cluster to the terminal cluster, N2.
Mentions: Currently, there is no high resolution structural information about mammalian complex I, but the structure of complex I from the thermophilic bacterium Thermus thermophilus has been described (5), and it contains the structures of the orthologs of all of the core subunits of the mammalian complex, including those of Nqo4 and Nqo6, the orthologs of the 49-kDa and PSST subunit, respectively. The sequences of Nqo4 and the 49-kDa subunit and Nqo6 and the PSST subunit are 61 and 64% conserved, respectively, with 42 and 48% identical, respectively, (supplemental Fig. S1), and therefore, the structure of complex I from T. thermophilus provides a reasonable model for examining the general environments of the modified arginine residues in the 49-kDa and PSST subunits of the bovine and human enzymes. A schematic representation of the region where the modified residues are found is shown in Fig. 9. The dimethylated Arg-85 in the bovine 49-kDa subunit replaces a threonine residue in the bacterial enzyme in a loop between β-strand 3 and α-helix 1, close to (∼7 Å) Fe-S cluster N2, which is attached to the PSST subunit; the larger side chain of the methylated arginine could be even closer. The methylation of an arginine residue increases the hydrophobicity and solvent-accessible surface of the side chain, reduces its potential to form hydrogen bonds, and lowers its pI value slightly (53). Cluster N2 is the terminal Fe-S cluster in the chain of seven Fe-S clusters, and if for example the methylated arginine residue were close to one of the side chains of the cysteine residues that ligand the cluster, the dimethylated arginine residue might conceivably influence its redox potential. Another possibility is that the methylated residue influences the assembly of complex I, and the association of human pathogenic mutations in putative protein methylases that lead to dysfunction of complex I (48) makes this an attractive possibility. The hydroxylated Arg-77 in the bovine PSST subunit is in a loop (not resolved in the structure of the bacterial complex) linking α-helices 2 and 3 close to the tunnel, also involving the ND1 and 49-kDa subunits, in which oxidized coenzyme Q is thought to bind so as to accept electrons from cluster N2. In the absence of more detailed structural information, it is currently not possible to understand the role of the hydroxylated arginine residue. It is unlikely that the hydroxylation of the arginine residue arises in the tunnel, for example by reaction with reactive oxygen species, as such species are not thought to be generated from the quinone (54), although this conclusion is disputed (55). The restricted access to both modified residues would make enzymatic modification via the tunnel unlikely, and therefore, it is much more probable that the modifications occur in the cytoplasm at any one of the stages following synthesis, during or following import into the mitochondrion or during assembly into complex I. The identification of the modifying enzymes is likely to help in determining the cellular site of modification, and it may help also in understanding the roles of the modified arginine residues.

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