Nitrogenase Activity

Acetylene-reduction assay Estimates nitrogenase activity by measuring the rate of acetylene reduced to ethylene. 

The pH dependence of nitrogenase activity has been interpreted in terms of a group with a pi a = 6.3 that must he deprotonated for activity and another group with a pi a = 9 that must be protonated for activity 128). The pi a of the latter group was moved about 0.5 pH units more acid in the presence of acetylene and carbon monoxide and the group with the pi of 6.3 was moved about 0.4 pH units more acid by acetylene. The behavior of the group with the pZa of 9 is fully consistent with earlier observations (50) on the effect of acetylene on... 

Crittenden, P.D. Kershaw, K.A. (1978). A procedure for the simultaneous measurement of net COj-exchange and nitrogenase activity in lichens. New... 

Barney, B.M., Lukoyanov, D., Yang, T.-C., Dean, D.R., Hoffman, B.M. and Seefeldt, L.C. (2006) A methyldiazene (NH=N-CH3)-derived species bound to the nitrogenase active-site FeMo cofactor implications for mechanism, Proc. Natl. Acad. Sci. U.S.A., 103, 17113-17118. 

In their 1960 paper, Carnahan et al. reported that ATP was inhibitory to nitrogenase activity in their cell-free preparations. Hence, when McNary and Burris [24] reported that ATP was needed to support fixation, the report was met with a good deal of skepticism. But experiments in a number of other laboratories verified the absolute need for ATP. Not only is ATP needed, it is needed in substantial amounts. Under ideal conditions 16 ATP are required per N2 reduced to 2 NH3. Under normal conditions in nature the requirement probably is in the 20 to 30 ATP per N2 range. N2 reduction is energy demanding whether it is accomplished chemically in the Haber process or enzymatically by the nitrogenase system. 

Growth inhibited Photosynthesis inhibited Nitrogenase activity inhibited... 

Fig. 19.25 Schematic diagram of nitrogenase activity in a bacterial cell. Carbohydrate provides reducing capacity (ferredoxin), energy (MgATP), and organic precursors for the manufacture of amino acids. [From Skinner. K. J. Chem. Eng. News 1976.54(41). 22-35. Reproduced with permission.]...

Nitrogenase catalyzes the reduction of a number of substrates in addition to N2 and H+. Acetylene is reduced to ethylene, and nitrous oxide to dinitrogen, while azide undergoes reduction to N2 and NH3. In the last two cases the product N2 is not reduced further, implying that it is not in the correct binding position for reduction. The presence of multiple sites on the MoFe protein suggested by this observation is also supported by the non-competitive nature of the inhibition shown by some of these compounds. The reduction of acetylene has been used as a marker reaction for nitrogenase activity. 

The development of the acetylene reduction assay to measure nitrogenase activity. 

Thus, the hybrid cluster is a putative iron-sulfur redox catalyst. It is, however, a very uncommon cluster (perhaps only comparable to the nitrogenase active site) in two aspects (1) it is a hybrid cluster i.e., it contains intrinsic building blocks that are distinctly strange to iron-sulfur clusters and (2) it can exist in more than two (in fact, four [63]) oxidation states. 

SCHEME 10.2 The cofactor for nitrogenase activity, an iron-sulfur-vanadium cluster. 

The X-ray structure of the nitrogenase active site is obviously a good starting point for investigations on mechanistic aspects of the... 

Newell, S.Y., Hopkinson, C.S., and Scott, L.A., (1992) Patterns of nitrogenase activity (acetylene reduction) associated with standing, decaying shoots of Spartina altemiflora. Estuar. Coastal Shelf Sci. 35, 127-140. 

Cyanobacteria are 02-evolving photosynthetic prokaryotes, many of which are able to fix atmospheric N2 via an ATP-dependent nitrogenase activity. The nitrogenase enzyme is oxygen-sensitive however, it is localized in specialized cells called heterocysts which lack PSII and has an envelope impermeable to 02. Under normal physiological conditions, the nitrogen fixed in the cells as ammonia is rapidly transformed to... 

Nitrogenases are very versatile enzymes. They reduce, in addition to N2, a lot of other substrates, for example, protons, acetylene, azide, nitriles, and isonitriles. All of these substrates are reduced by multiples of [2 H+/2 e ] reductions. Both CO and NO inhibit nitrogenase activity. The apparent [2 H+/2 e ] multiplicity of substrate reductions and a couple of other findings strongly suggest diazene and hydrazine to be intermediates of the N2 —> NH3 reduction. 

---