Abstract
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+III2+IV (S1) 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 39kDa (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.
Highlights
Production of energy in most aerobic cells is provided by the joint activity of the mitochondrial respiratory chain and ATP-synthase
Complex I dihydronicotinamide adenine dinucleotide (NADH):Q1 oxidoreductase activity was assessed: 25 μg submitochondrial particles (SMP) were diluted in 1 ml PBS pH 7.5 supplemented with 10 μM NADH to convert the enzyme to the A-form. 1 mM KCN, 30 μM Q1 and 165 μM NADH were added to the suspension and the NADH oxidation rate was measured in the linear part of the curve
As observed after silver staining (Fig. 1C), the composition of the supercomplexes containing A and D forms of complex I was similar. Taken together these results suggest that the active/de-active transition (A/D) conformational change(s) of complex I has no effect on the association of the enzyme with other respiratory chain complexes in vitro as revealed by native electrophoresis
Summary
Production of energy in most aerobic cells is provided by the joint activity of the mitochondrial respiratory chain and ATP-synthase. Recent studies [3,4] have significantly improved our understanding of the structure, the proton translocation machinery [4,5] and assembly of complex I [6] but many aspects of the enzyme's regulation require elucidation
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Biochimica et Biophysica Acta (BBA) - Bioenergetics
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
