Binding of 14-3-3 to P-NR has been shown to interrupt whole-chain electron transfer from NADH to nitrate, and the partial activity from the electron donor methylviologen (MV) to nitrate was also blocked (Bachmann et al. 1996a). Most probably, 14-3-3-binding causes a conformational change which inhibits electron transfer from the heme-Fe of the Cyt b moiety to MoMPT. That conformation change may prevent the cationic MV electron donor to interact. Interestingly, the Km values for NADH (1 to 7 pM) and nitrate (20 to 40 pM) are not changed by the above inactivation mechanism (Kaiser and Spill 1991). Thus, phosphorylation and 14-3-3 binding do not change kinetic properties of the enzyme, but only the ratios of free NR (active) to P-NR-14-3-3 complexes (inactive). Neither in leaves nor in roots was NR activity ever completely abolished or fully activated by the above modulation. In fact, as a general rule, a very low activation state in darkened leaves is about 20% (of total NR activity measured with an excess of EDTA), and a maximum activation state in illuminated leaves supplied with high CO2 is 80%.
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