Specific Methylation of Asp160 (49 kDa subunit) Located inside the Quinone Binding Cavity of Bovine Mitochondrial Complex I.

Abstract

Asp160 in the 49 kDa subunit of bovine mitochondrial complex I, which is located in the inner part of the quinone binding cavity, is considered to be an essential residue for energy conversion of the enzyme. To elucidate the catalytic function of this residue, we attempted to specifically methylate 49 kDa Asp160 [Asp(COO)-CH3] through a ligand-directed tosyl (LDT) chemistry technique with an acetogenin derivative (ALM) as a high-affinity ligand. We confirmed the specific methylation of 49 kDa Asp160 through liquid chromatography-tandem mass spectrometry analysis of the tryptic digests of the 49 kDa subunit. The binding affinity of a quinazoline-type inhibitor ([(125)I]AzQ) occupying the quinone binding cavity was not affected by methylation, indicating that this chemical modification does not induce significant structural changes inside the quinone binding cavity. The methylation of 49 kDa Asp160 did not lead to the complete loss of catalytic activity; the modified enzyme retained partial electron transfer and proton translocation activities. These results along with the fact that 49 kDa Asp160 elicits a very strong nucleophilicity against various LDT reagents in the local protein environment strongly suggest that this residue is free from strict interactions (such as electrostatic interaction) arising from nearby residue(s) and is functionally important but not essential for the energy conversion of complex I.