Native organization of alternative NAD(P)H-dehydrogenases NDA and NDB in mitochondria of etiolated pea sprouts

Numerous biochemical and structural studies into the native organization of oxidative phosphorylation in the mitochondria of various eukaryotic organisms have convincingly shown that respiratory complexes can associate with one another to form higher-order structures referred to as supercomplexes. Plant mitochondria are distinguished by a more complicated organization of the respiratory chain due to the presence of a number of alternative oxidoreductases. It is considered that these enzymes do not physically interact with those of the cytochrome pathway. However, the available literature data obtained on yeast mitochondria suggests the possibility of such an association. In this regard, we aimed to study the native organization of alternative NAD(P)H-dehydrogenases NDA and NDB in plant mitochondria. The work was performed on six-day etiolated pea seedlings. The 2D BN/SDS-PAGE in combination with immunochemistry found that, in pea organelles, the main part of the populations of NDA and NDB alternative NAD(P)H dehydrogenases were included in superstructures with masses of 700, 780, and 900 kDa. Additionally, NDA was detected in the region of 1480 and 1600 kDa, and NDB was registered at values of 1330, 340, and 100–120 kDa. An analysis of subunit profiles of the observed associations and a colorimetric detection of ATPase activity in 1D BN-gel suggested that the major part of the NDA and NDB populations identified by the available antibodies was associated with ATP synthase and represented a heterogeneous population of ATP-synthasomes, assumably, with a NDA 2 /NDB 2 Va/b 1-2 composition. The rest of the enzymes were likely to be part of the NDA 2 /NDB 2 III 2 IV and NDA 2 IV 1 Va 2 supercomplexes. The physiological significance of the association of alternative NAD(P)H dehydrogenases with ATP synthase requires further study.