Direct measurements of steady-state kinetics of cyanobacterial n(2) uptake by membrane-leak mass spectrometry and comparisons between nitrogen fixation and acetylene reduction.

A mass spectrometer with a membrane-covered inlet was used to measure nitrogen fixation by following changes in the concentration of dissolved N(2) in a stirred suspension of the cyanobacterium Anabaena variabilis in an open system. The results showed a good fit to Michaelis-Menten kinetics with a K(m) for N(2) of 65 muM at 35 degrees C, corresponding to 0.121 atmosphere of N(2). Corresponding values for the K(m) for acetylene reduction were 385 muM (0.011 atmosphere at 35 degrees C). Comparison of the values of V(max) for N(2) uptake with those for the acetylene reduction assay under similar conditions gave an average value of 3.8 for the conversion factor between N(2) and C(2)H(2) reduction. Reduction of protons to hydrogen was completely inhibited at sufficiently high concentrations of C(2)H(2), but even at saturating N(2) concentrations, 1 mol of H(2) was produced for every mole of N(2) reduced. This explains the finding that the observed C(2)H(2)/N(2) ratio is higher than the value of 3 expected from the requirement for two electrons for acetylene reduction and six for nitrogen reduction. The results correlate well with a mechanism for N(2) reduction involving the equation: N(2) + 8H + 8e --> 2NH(3) + H(2) which gives a conversion factor between C(2)H(2) and N(2) of 4. It is proposed that, in general, 4 is a more appropriate value than 3 for the conversion factor.