In living organisms, respiration is a biological process degrading different carbon substrates, consuming O2, and releasing the carbon as CO2. Plants have several alternative enzymes that are involved in the respiratory processes, as compared to animals. These alternative respiratory enzymes allow electrons to be transferred to oxygen in the mitochondrial inner membrane, but bypassing ATP synthesis. The alternative enzymes, e.g., type II NAD(P)H dehydrogenases (NDH-2), affect cellular NAD(P)H redox status, which is of vital importance for energy metabolism, ROS production and removal, anti-oxidation and reductive biosynthesis.
Plant NDB-type proteins are NDH-2 enzymes located at the external mitochondrial inner membrane. It was earlier found that NDB1 oxidise cytosolic NADPH, and NDB2 oxidise cytosolic NADH. In this study, the regulatory mechanisms of Arabidopsis thaliana and Solanum tuberosum NDB1 by cytosolic Ca2+ and pH were studied and compared to NDB2, using purified mitochondria and E. coli-produced proteins in a membrane-bound and a purified soluble state. Membrane bound NDB1 and NDB2 oxidised NADPH and NADH, respectively. Soluble forms of NDB1 oxidise both NADH and NADPH, with higher NADPH activity. Soluble forms of NDB2 oxidised only NADH like the membrane-bound enzyme. In solution, the active StNDB1 resided as oligomers of dimeric units, mainly hexamers, and recombinant AtNDB2 was highly oligomeric. Within a physiological pH range, an acidic pH was found to lower the Ca2+ demand for activation of the mitochondrial and E. coli-produced NADPH oxidation via NDB1, as compared to a more alkaline pH. Depending on pH, 3-82 µM Ca2+ was needed. In contrast, the sub-µM Ca2+ demand for activation of NADH oxidation was not linked to pH. Both soluble and mitochondrial StNDB1 (NADPH oxidation) could respond quickly to increased and decreased Ca2+, whereas mitochondrial NADH oxidation responded quickly to Ca2+ increase but slowly to Ca2+ decrease. Overall, the results suggest that in vivo, the activity of NDB1 is rapidly controlled by pH-shift-associated Ca2+ spikes in the cytosol whereas NDB2 may be more continuously active.
Based on modelling of NDB1, the core catalytic parts and dimerization surface showed distinct similarities to the structures of yeast ScNDI1 and Plasmodium falciparum PfNDH-2. This analysis highlighted motifs that correlate with NAD(P)H substrate specificity, and which were followed by evolutionary analysis. Most eukaryotic species have NDB proteins that contain a non-acidic motif for NADPH binding. Angiosperms and liverworts contain NDB proteins of NDB1- and NDB2- type, i.e. they contain acidic and non-acidic motifs for NADH and NADPH binding, respectively. This indicates that plants have more flexibility for external NAD(P)H oxidation as compared to other eukaryotes. Based on the evolutionary analysis, Ca2+-dependent external NADPH oxidation appears to be an ancient process as compared to NADH oxidation, and thus possibly has a more fundamental function in cellular redox metabolism.
- Rasmusson, Allan, Supervisor
- Hederstedt, Lars, Assistant supervisor
|Award date||2019 May 29|
|Place of Publication||Lund|
|ISBN (electronic) ||978-91-7753-975-9 |
|Publication status||Published - 2019 May 3|
Place: Biology lecture hall (A213), the Biology building, Sölvegatan 35, Lund
Name: Hamborg Nielsen, Tom
Title: Associate Professor
Affiliation: Copenhagen University, Copenhagen, Denmark
- Electron transport
- Plant mitochondria
- Type II NAD(P)H dehydrogenase