NDUFAF7 Methylates Arginine 85 in the NDUFS2 Subunit of Human Complex I

Virginie F Rhein,Joe Carroll,Shujing Ding,Ian M Fearnley,John E Walker

doi:10.1074/jbc.m113.518803

Virginie F Rhein, Joe Carroll + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m113.518803

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Abstract

Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 subunits. One arm is embedded in the inner membrane with the other protruding ∼100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH and the primary electron acceptor FMN, and it provides a scaffold for seven iron-sulfur clusters that form an electron pathway linking FMN to the terminal electron acceptor, ubiquinone, which is bound in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, probably energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Complex I is put together from preassembled subcomplexes. Their compositions have been characterized partially, and at least 12 extrinsic assembly factor proteins are required for the assembly of the complex. One such factor, NDUFAF7, is predicted to belong to the family of S-adenosylmethionine-dependent methyltransferases characterized by the presence in their structures of a seven-β-strand protein fold. In the present study, the presence of NDUFAF7 in the mitochondrial matrix has been confirmed, and it has been demonstrated that it is a protein methylase that symmetrically dimethylates the ω-N(G),N(G') atoms of residue Arg-85 in the NDUFS2 subunit of complex I. This methylation step occurs early in the assembly of complex I and probably stabilizes a 400-kDa subcomplex that forms the initial nucleus of the peripheral arm and its juncture with the membrane arm.

Highlights

Arginine 85 of NDUFS2, a subunit of mitochondrial complex I, is symmetrically dimethylated
The extrinsic arm contains binding sites for NADH and the primary electron acceptor FMN, and it provides a scaffold for seven iron-sulfur clusters that form an electron pathway linking FMN to the terminal electron acceptor, ubiquinone, which is bound in the region of the junction between the arms
Complex I is put together from preassembled subcomplexes. Their compositions have been characterized partially, and at least 12 extrinsic assembly factor proteins are required for the assembly of the complex

Summary

Conclusion

Methylation of arginine 85 of NDUFS2 is required for assembly of complex I. The presence of NDUFAF7 in the mitochondrial matrix has been confirmed, and it has been demonstrated that it is a protein methylase that symmetrically dimethylates the ␻-NG,NGⴕ atoms of residue Arg-85 in the NDUFS2 subunit of complex I This methylation step occurs early in the assembly of complex I and probably stabilizes a 400kDa subcomplex that forms the initial nucleus of the peripheral arm and its juncture with the membrane arm. This intermediate complex is expanded by the addition of the distal region of the membrane arm, a domain containing the NADH and FMN binding sites to the distal region of the matrix arm, and the remaining supernumerary subunits [16] This assembly process depends upon the participation of at least 12 exogenous protein factors that are not constituents of the mature functional enzyme. We have shown recently that this residue carries two methyl groups attached symmetrically to the ␻-NG- and ␻-NG⬘-nitrogen atoms of its guanidino group [37]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION