Nitrogenase proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium.
Gluconacetobacter diazotrophicus Pal-5 grew well and expressed nitrogenase activity in the absence of NH4+ and at initial O2 concentrations greater than 5% in the culture atmosphere. G. diazotrophicus nitrogenase consisted of two components, Gd1 and Gd2, which were difficult to separate but were purified individually to homogeneity. Their compositions were very similar to those of Azotobacter vinelandii nitrogenase, however, all subunits were slightly smaller in size. The purified Gd1 protein contained a 12:1 Fe/Mo ratio as compared to 14:1 found for Av1 purified in parallel. Both Gd2 and Av2 contained 3.9 Fe atoms per molecule. Dithionite-reduced Gd1 exhibited EPR features at g=3.69, 3.96, and 4.16 compared with 3.64 and 4.27 for Av1. Gd2 gave an S=1/2 EPR signal identical to that of Av2. A Gd1 maximum specific activity of 1600 nmol H2 (min mg of protein)(-1) was obtained when complemented with either Gd2 or Av2, however, more Av2 was required. Gd2 had specific activities of 600 and 1100 nmol H2 (min mg protein)(-1) when complemented with Av1 and Gd1, respectively. The purified G. diazotrophicus nitrogenase exhibited a narrowed pH range for effective catalysis compared to the A. vinelandii nitrogenase, however, both exhibited maximum specific activity at about pH 7. The Gd-nitrogenase was more sensitive to ionic strength than the Av-nitrogenase.
Fisher
Karl
K
Department of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Newton
William E
WE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism / Q000494:pharmacology; D016948:Azotobacter vinelandii / Q000201:enzymology; D002248:Carbon Monoxide / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D041821:Gluconacetobacter / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009586:Nitrogen Fixation / Q000187:drug effects; D009591:Nitrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D031786:Saccharum / Q000382:microbiology*; D012965:Sodium Chloride / Q000494:pharmacology; D017186:Titrimetry
D012965:Sodium Chloride; D000641:Ammonia; D002248:Carbon Monoxide; D006859:Hydrogen; D000255:Adenosine Triphosphate; D009591:Nitrogenase; D010100:Oxygen