THE MOLYBDENUM AND VANADIUM NITROGENASES OF AZOTOBACTER-CHROOCOCCUM - EFFECT OF ELEVATED-TEMPERATURE ON N2 REDUCTION

During the reduction of N, by V-nitrogenase at 30-degrees-C, some hydrazine (N2H4) is formed as a product in addition to NH3 [Dilworth and Eady (I 99 1) Biochem. J. 277, 465-468]. We show here the following. (1) That over the temperature range 30-45-degrees-C the apparent K(m) 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 H-2; 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 H-2 evolution and N2 reduced to NH3 at N2 pressures up to 339 kPa for both Mo- and V-nitrogenases gave limiting H-2/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 Buff is [(l 984) Science 224, 1095-1097] at 5650 kPa, there appears to be little or no divergence from the predictions based on the apparent K(m) 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.