Specific Methylation of Asp160 (49 kDa subunit) Located inside the Quinone Binding Cavity of Bovine Mitochondrial Complex I (original) (raw)
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Biochemistry, 2017
We previously showed that a bulky ring-strained cycloalkyne possessing a rhodamine fluorophore directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine heart submitochondrial particles [Masuya, T., et al. (2014) Biochemistry 53, 7816-7823]. This two-step conjugation may be a promising technique for specific chemical modifications of the quinone-access channel in complex I by various molecular probes, which would lead to new methodologies for studying the enzyme. However, because the reactivities of ring-strained cycloalkynes are generally high, they also react with other nucleophilic amino acids in mitochondrial proteins, resulting in significant undesired side reactions. To minimize side reactions and achieve precise pinpoint chemical modification of 49 kDa Asp160, we investigated an optimal pair of chemical tags for the two-step conjugation reaction. We fou...
Exploring the binding site of acetogenin in the ND1 subunit of bovine mitochondrial complex I
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
125 I-labeled (trifluoromethyl)phenyldiazirinyl acetogenin, [ 125 I]TDA, a photoaffinity labeling probe of acetogenin, photo-cross-links to the ND1 subunit of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) with high specificity [ M. Murai, A. Ishihara, T. Nishioka, T. Yagi, and H. Miyoshi, (2007) The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I, Biochemistry 46 6409-6416.]. To identify the binding site of [ 125 I]TDA in the ND1 subunit, we carried out limited proteolysis of the subunit cross-linked by [ 125 I]TDA using various proteases and carefully analyzed the fragmentation patterns. Our results revealed that the cross-linked residue is located within the region of the 4th to 5th transmembrane helices (Val144-Glu192) of the subunit. It is worth noting that an excess amount of shortchain ubiquinones such as ubiquinone-2 (Q 2) and 2-azido-Q 2 suppressed the cross-linking by [ 125 I]TDA in a concentration-dependent way. Although the question of whether the binding sites for ubiquinone and different inhibitors in complex I are identical remains to be answered, the present study provided, for the first time, direct evidence that an inhibitor (acetogenin) and ubiquinone competitively bind to the enzyme. Considering the present results along with earlier photoaffinity labeling studies, we propose that not all inhibitors acting at the terminal electron transfer step of complex I necessarily bind to the ubiquinone binding site itself.
Properties of the active site lysyl residue of mitochondrial aspartate aminotransferase in solution
The Journal of biological chemistry, 1983
Two vitamin B6 derivatives, N-bromoacetylpyridoxamine (BAPM) and its phosphate ester have been found to be affinity-labeling reagents for mitochondrial aspartate aminotransferase (EC 2.6.1.1). These derivatives were first shown to react with a critical sulfhydryl group in tryptophan synthase (Higgins, W., and Miles, E. W. (1978) J. Biol. Chem. 253, 4648-4652). In the apoaminotransferase, BAPM has now been found to inactivate by covalently modifying a critical lysyl residue, preventing reconstitution of the apoenzyme by pyridoxal 5'-phosphate. The dependence of the rate of inactivation upon the concentration of the reagent is consistent with a rapid equilibrium binary complex formation prior to the inactivation reaction. Both the dissociation constant for this complex and the rate of the reaction leading to inactivation are dependent on pH. BAPM binds best from pH 7.5 to 8.5. The rate of inactivation increases from pH 6 to 9. Succinate and phosphate competitively bind to the apoe...
ND3, ND1 and 39kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III 2 + IV (S 1 ) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.
Chemical Research in Toxicology, 2005
The reaction of cytochrome c with the electrophilic compounds (glutathion-S-yl)-1,4-benzoquinone (GSBQ) and iodoacetamide was studied using mass spectrometry. GSBQ is a nephrotoxic quinol-thioether metabolite of benzoquinone, while iodoacetamide is an alkylating agent targeting cysteine thiols. Both chemicals formed covalent adducts with cytochrome c. GSBQ formed adducts with cytochrome c at pH 6 on several histidine and lysine residues. At a pH >7, the initial product rearranged to a disubstituted cyclic quinone species preferentially found at two sites on the protein, Lys25-Lys27 and Lys86-Lys87, via quinol amine linkages. These two sites were previously determined to be the targets of benzoquinone adduct formation [Person et al. (2003) Chem. Res. Toxicol. 16, 598-608]. Cyclic reaction products are preferentially formed at two sites on the protein because of the presence of multiple basic residues in a conformationally flexible region whereas noncyclic products bind to a broad spectrum of available lysine and histidine nucleophiles. Iodoacetamide was a less selective alkylating agent able to form adducts on the majority of the nucleophilic sites of the protein. MS/MS spectra were used to identify signature ions for GSBQ-adducted peptides from the characteristic fragmentation patterns. Neutral losses of the 129 Da gamma-glutamate residue and of the 273 Da glutathione moiety were found in both cysteine thiol- and lysine amine-linked GSBQ adduct MS/MS. Characteristic fragment ions were used in conjunction with the scoring algorithm for spectral analysis to search for adducted species present at low levels in the sample, and the analysis is applicable generally to detection of glutathione conjugates by MS/MS. Parallel analysis using matrix-assisted laser desorption/ionization-MS to compare spectra of control and treated samples allowed identification of peptide adducts formed by direct addition of GSBQ and by the subsequent loss of the glutathione moiety in a pH-dependent cyclization reaction.