Direct Measurements of Steady-State Kinetics of Cyanobacterial N 2 Uptake by Membrane-Leak Mass Spectrometry and Comparisons Between Nitrogen Fixation and Acetylene Reduction

Abstract

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 μM at 35°C, corresponding to 0.121 atmosphere of N 2 . Corresponding values for the K m for acetylene reduction were 385 μM (0.011 atmosphere at 35°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.