The molybdenum and vanadium nitrogenases of Azotobacter chroococcum: effect of elevated temperature on N2 reduction

During the reduction of N2 by V-nitrogenase at 30 degrees C, some hydrazine (N2H4) is formed as a product in addition to NH3 [Dilworth and Eady (1991) Biochem. J. 277, 465-468]. We show here the following. (1) That over the temperature range 30-45 degrees C the apparent Km for the reduction of N2 to yield these products is the same, but increases from 30 to 58 kPa of N2. On increasing the temperature from 45 degrees C to 50 degrees C, little change occurred in the rate of reduction of protons to H2; the rate of N2H4 production increased, but the rate of NH3 formation decreased 7-fold. (2) Temperature-shift experiments from 42 to 50 degrees C or from 50 to 42 degrees C showed that this selective loss of the ability to reduce N2 to NH3 was reversible. The effects we observe are consistent with the existence of different conformers of the VFe-protein at the two temperatures, that predominating at 50 degrees C being largely unable to reduce N2 to ammonia. (3) Measurement of the ratio between H2 evolution and N2 reduced to NH3 at N2 pressures up to 339 kPa for both Mo- and V-nitrogenases gave limiting H2/N2 values of 1.13 +/- 0.13 for Mo-nitrogenase and 3.50 +/- 0.03 for V-nitrogenase. Since for Mo-nitrogenase our measured value for the ratio at 339 kPa is the same as that derived by Simpson and Burris [(1984) Science 224, 1095-1097] at 5650 kPa, there appears to be little or no divergence from the predictions based on the apparent Km for N2. These data then suggest that there may be a fundamentally different mechanism for N2 binding to V-nitrogenase compared with Mo-nitrogenase. (4) We did not detect any N2H4 as a product of N2 reduction by Mo-nitrogenase over the temperature range investigated; however, at 50 degrees C this system reduced acetylene (C2H2) to yield some ethane (C2H6), in addition to ethylene (C2H4), a reaction normally associated with Mo-independent nitrogenases.