Characterising structural and functional differences between catalytically active and de-active forms of mammalian mitochondrial complex I

Abstract

In some organisms complex I (NADH dehydrogenase/NADH ubiquinone oxidoreductase) is thought to adopt two catalytically distinct states, an ‘active’ (A) or 'deactive' (D) form. Complex I is believed to convert to an ‘idle’ D-form when incubated at 30-37°C in the absence of substrate NADH (nicotinamide adenine dinucleotide) or if electron acceptor ubiquinone is fully reduced. The D-form of complex 1 is thought to be ‘re-activated’ following turnover in the presence of NADH. At alkaline pH, in the presence of divalent cations or free fatty acids an increased number of turnovers with NADH is required for re-activation. These observations of mammalian mitochondrial complex 1 were used to characterise the enzyme in different preparations of bovine heart, rat brain and mouse heart and brain.

A cross-linking approach was applied to examine the proximity of subunits and determine any conformational differences between active and de-active forms of complex I from bovine heart submitochondrial particles (SMPs). The cross-linking reagents; EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline), EMCS (N-(e-maleimidocaproyloxy)succinimide ester, Sulpho-EMCS (N-(e-maleimidocaproyloxy)sulphosuccinimide ester), DSS (disuccinimidyl suberate) and DSP (dithiobis(succinimidyl propionate) were selected for their different chemical specificities and spacer arm lengths varying from zero- to 12 A. Complex I was isolated by BN-PAGE (Blue native polyacrylamide gel electrophoresis) and subunits were separated by dSDS-PAGE (double SDS polyacrylamide gel electrophoresis) for silver staining.

The same cross-linked products with cytochrome c oxidase (complex IV) were found with crosslinkers EEDQ and EMCS irrespective of whether complex 1 was treated for the predominantly active- or the predominantly de-active form. Complex IV subunit I, subunit 4 isoform I and subunit 7A-related protein formed cross-linked products with complex I treated with zero and 9.4 A cross-linking reagents, suggesting a close association of complex I and IV. Complex I also formed cross-linked products with complex II (succinate dehydrogenase/succinate ubiquinone oxidoreductase) subunit II, and an unidentified target, the significance of which, remains to be determined.

Active and de-active form complex I rat brain SMPs were treated with the S- nitrosothiols: GSNO (S-nitrosoglutathione) and SNAP (S-nitroso-N-acetylpenicillamine). Any sites of S-nitrosation that differed between the two forms of complex I were determined by differences in fluorescent intensity following a DIGE (difference gel electrophoresis) approach.

Young and old mouse heart and brain were isolated by a simple membrane isolation technique. Significant differences in complex 1 and complex IV activity were determined with age.

Date of Award	Jul 2019
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Alexander Galkin (Supervisor) & David J. Timson (Supervisor)