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  • The presence of external NAD P


    The presence of external NAD(P)H dehydrogenases (NDE) on the outer surface of the inner mitochondrial membrane enables the mitochondria to oxidize cytosolic NAD(P)H (Moller 2001). A porin (a voltage-dependent anion-selective channel, VDAC) that is abundant in the outer mitochondrial membrane permits the passage of molecules of <5kDa and thus ionically and metabolically connects the intermembrane space to the cytosol. A substantial amount of evidence, primarily in plants and fungi, points to the existence of two separate Apocynin oxidizing either cytosolic NADH or cytosolic NADPH in the inner mitochondrial membrane (Carneiro et al., 2004, Carneiro et al., 2007, Fredlund et al., 1991, Luttik et al., 1998, Melo et al., 2001, Michalecka et al., 2004, Rigoulet et al., 2004, Roberts et al., 1995). Specifically, NADPH oxidation is more sensitive to inhibition by diphenyleneiodonium chloride (IDP) than NADH. However, because diphenyleneiodonium chloride is not sufficiently specific, it substantially complicates the research (Kerscher, 2000, Rasmusson et al., 2004). Some alternative dehydrogenases contain a sequence with a more or less degenerate EF-hand motifs that indicates Ca2+ binding (Michalecka et al., 2003, Rasmusson and Moller, 1991). In plant mitochondria, Ca2+ dependence has been observed for external NADH and NADPH oxidation (Moller and Palmer, 1981, Moller et al., 1982, Rasmusson et al., 2004) with NADH oxidation being less sensitive (Arron and Edwards 1980). Moreover, NADPH oxidation appears to require more Ca2+ for activity than NADH oxidation, which could strengthen the possibility that there are two separate enzymes for NADH and NADPH. Interestingly, in most cases, the NADH-oxidizing enzymes can also oxidize NADPH due to the structural similarity of both molecules that differ in the presence of a phosphate group in NADPH. However, in alkaline pH, the oxidation of NADPH could be prevented by electrostatic repulsion between the negative charges at the phosphate group of NADPH and the phospholipids of the membrane (Moller and Palmer 1981). There are also reports that demonstrate the dependence of the internal NAD(P)H dehydrogenases (NDI) on calcium ions (Rasmusson and Moller 1991). The calcium effect is thought to be mediated by an electrostatic screening of the negatively charged phospholipid membrane, which allows for the negatively charged NAD(P)H molecule to approach the membrane, thus increasing the affinity for NAD(P)H (Johnston et al. 1979). Because alternative dehydrogenases exhibit maximal activity in acidic pH values, this attribute seems to play a significant role in providing the proper activity of these enzymes at the physiological pH and/or under stress conditions. In addition, changes in the pH and Ca2+ may differentially regulate NAD(P)H oxidation and therefore affect the cellular redox state (Rasmusson et al. 2008). Recently, it has been proposed that EF-hand motifs may constitute sites for regulation of enzyme activity by acting on the residues that stabilize/protonate FAD in different oxidation states during enzymatic reaction (Marreiros et al. 2017). The number, substrate specificity, and Ca2+ dependence of alternative NAD(P)H dehydrogenases vary significantly when different organisms are compared. For instance, Arabidopsis mitochondria may possess up to seven proteins, including three external dehydrogenases: two are NADH dehydrogenases, one of which is calcium-dependent the other is calcium-independent, and the third dehydrogenase is a Ca2+-dependent NADPH enzyme (Geisler et al. 2007). The other three enzymes are confirmed to locate towards the matrix side, and one, NDC1, has also been found in plastoglobules (Ytterberg et al. 2006). When examining the sequence homology to type II NADH dehydrogenases in yeast and E. coli, only two genes, nda1 and ndb1, were found in potato. Based on the results of the protein import into the mitochondria, the NDA1 and NDB1 proteins have been determined to be directed to the inner and outer surface of the inner mitochondrial membrane, respectively (Rasmusson et al. 1999).