Evidence for the presence of two rotenone-insensitive NAD(P)H dehydrogenases on the inner surface of the inner membrane of potato tuber mitochondria

Submitochondrial particles were isolated from potato (Solanum tuberosum L.) tubers. The latency of cytochrome-c oxidase and succinate dehydrogenase indicated that they were 90% inside-out. The inability of the submitochondrial particles to form a membrane potential inside negative as monitored by safranine absorbance changes and their ability to form a large membrane potential inside positive as monitored by oxonol VI absorbance changes confirmed this sidedness. Through the use of rotenone to inhibit Complex I, and diphenyleneiodonium to inhibit both Complex I (by binding to the FMN in the active site) as well as rotenone-insensitive NADPH oxidation, it was possible to distinguish three different NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane: (1) a rotenone-sensitive, diphenyleneiodonium-sensitive, Ca²⁺-independent enzyme which prefers NADH as the substrate, i.e., Complex I; (2) a rotenone-insensitive, diphenyleneiodonium-sensitive, Ca²⁺-dependent NAD(P)H dehydrogenase; (3) a rotenone-insensitive, diphenyleneiodonium-insensitive, Ca²⁺-independent NADH dehydrogenase. All three enzymes were linked to the electron transport chain before Complex III as shown by antimycin A sensitivity and to proton pumping as shown by the generation of a membrane potential. The possible significance of these three enzymes for the function of the mitochondrion in the plant cell is discussed.