INHIBITION OF NITROGEN FIXATION BY OTHER GASES 293 



Inhibition of Azotohacter growth by N2O occurs if N2 is the sole source 

 of nitrogen, but does not if ammonia (Repaske and Wilson, 1952) or nitrate 

 (Mozen et al., 1955) is present, indicating that the site of the block is prev- 

 ious to these substances. Likewise, in Clostridium H^ inhibits uptake of 

 N2 but not of ammonia (Hiai et al., 1957). These inhibiting gases are oc- 

 casionally utilized. For example, NgO is assimilated by Azotohacter, although 

 slowly, and this is inhibited by N, and Hg (Burris, 1956). It is quite likely 

 that the competition in all these cases, with the possible exception of Og, 

 is at the nitrogenase active site binding Ng. Nitrogenase is a metalloflavo- 

 protein containing molybdenum and it is reasonable that these gases are 

 bound to the metal, the catalysis of Ng reduction being of similar type to 

 those mediated by various inorganic metal preparations (which are also 

 inhibited by other gases). 



It will be necessary before discussing mechanisms of inhibition in greater 

 detail to consider the enzyme hydrogenase, which has recently been closely 

 linked to nitrogen fixation, and its inhibitions. This enzyme catalyzes the 

 reduction of some unknown primary acceptor by molecular Ho and the 

 reduced acceptor then transfers the hydrogen atoms to other acceptors, 

 such as dyes, NAD, or eventually oxygen. It has been postulated that it 

 may in some instances participate in the reduction of Ng. The inhibition 

 by O2 is primarily due to oxygenation of the enzyme: 



E + nO, ^ E(02)„ 



and this inhibition is reversible upon removal of the O2 by dialysis (Krasna 

 and Rittenberg, 1954; Fisher et al., 1954). Prolonged exposures to Og 

 lead to progressive inactivation of bacterial hydrogenase (Shug et al., 

 1956). Although n has generally been assumed to be 1, Atkinson (1956) 

 obtained rather complex data possibly indicating a value of 2 for the 

 Hydrogenomonas facilis enzyme. The hydrogenase-catalyzed evolution of 

 H2 in Rhodospirillum rubrum (Lindstrom et al, 1949) and soybean root 

 nodules (Hoch et al, 1960) is inhibited by Ng. Although this might be at- 

 tributed in part to a diversion of the flow of hydrogen atoms to the reduction 

 of N2, Bregoff and Kamen (1952) observed that 1 mole of N2 prevents 

 the release of several moles of Hg. One of the difficulties in assuming a direct 

 competition between Hg and Ng for the hydrogenase active site is the 

 fact that, despite the inhibition of Hg evolution by N2, the exchange reac- 

 tion whereby HD is formed from D2 and a hydrogen donor is actually accel- 

 erated by N2 (Hoch et al., 1960). The Hj inhibition of nitrogen fixation 

 was previously claimed to be competitive, but Parker and Dilworth (1963) 

 found that Hg causes a lag in the N2 uptake at low pN2, whereas in cells 

 of Azotohacter vinelandii adapted to Hg the lag is abolished. Taking the 

 lag phase into account, the inhibition is not competitive; from the reciprocal 



