Direct Measurements of Steady-State Kinetics of Cyanobacterial N2 Uptake by Membrane-Leak Mass Spectrometry and Comparisons Between Nitrogen Fixation and Acetylene Reduction

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A mass spectrometer with a membrane-covered inlet was used to measure nitrogen fixation by following changes in the concentration of dissolved N2 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 Km for N2 of 65 μM at 35°C, corresponding to 0.121 atmosphere of N2. Corresponding values for the Km for acetylene reduction were 385 μM (0.011 atmosphere at 35°C). Comparison of the values of Vmax for N2 uptake with those for the acetylene reduction assay under similar conditions gave an average value of 3.8 for the conversion factor between N2 and C2H2 reduction. Reduction of protons to hydrogen was completely inhibited at sufficiently high concentrations of C2H2, but even at saturating N2 concentrations, 1 mol of H2 was produced for every mole of N2 reduced. This explains the finding that the observed C2H2/N2 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 N2 reduction involving the equation: N2 + 8H+ + 8e− → 2NH3 + H2 which gives a conversion factor between C2H2 and N2 of 4. It is proposed that, in general, 4 is a more appropriate value than 3 for the conversion factor.

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