L.A. Syrtsova, V.A. Nadtochenko, N.N. Denisov, E.A. Timofeeva, N.I. Shkondina V.Yu. Gak
Institute of Problems of Chemical Physics of Russian Academy of Sciences, Chernogolovka, Moscow region, 142432 Russia
The kinetics of minimum turnover of nitrogenase, transfer of two electrons from a photodonor (a system containing eosin and NADH or 4,5 -dibromofluorescein and NADH) to Fe protein (Av2) and the kinetics of transfer of the first and second electrons from Av2 to MoFe protein (Avl) were studied by kinetic laser spectroscopy. The effects of the substrates of nitrogenase (nitrogen, acetylene, and protons) on the intramolecular electron transfer in nitrogenase were studied. Analysis of the effect of photodonor excitation radiation intensity on the rate of electron transfer was used to determine the transfer rate constants for the first (fe) and second (fe) electrons from Av2 to Avl. In the presence of MgATP two electrons are sequentially transferred from Av2 to Avl, and no delay between these reactions was detected. The first electron transferred from Av2 to Avl is not targeted to the substrate; fe = 154 ± 15 sec"1 at 23°C for the system 4,5-dibromofluorescein-NADH; fe = 53 ± 5 sec"1, 95 ± 9 sec"1, and 24 ± 2 sec"1 at 23°C in the presence of nitrogen, acetylene, and argon, respectively. An unidentified slow step (fe = 18 ± 2 sec"1 at 23°C) may be associated with electron transfer within Avl.
Steps of nitrogenase reaction with photodonor DBF-NADH presence of nitrogen:
The little circle is Av2, large circle is Avl in reduced states.
and super-reduced k0= 200c"1, ki= 158 c"1, k2 = 54c"1, k3 = 18 c"1
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