Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.
The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)
Hyman
M R
MR
Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis 97330-2902.
Seefeldt
L C
LC
Morgan
T V
TV
Arp
D J
DJ
Mortenson
L E
LE
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D016948:Azotobacter vinelandii / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009569:Nitric Oxide / Q000494:pharmacology*; D009591:Nitrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013053:Spectrophotometry
D046911:Macromolecular Substances; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007501:Iron; D009591:Nitrogenase