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Appl Environ Microbiol. 1983 April; 45(4): 1331-1337
Copyright © 1983, American Society for Microbiology. All Rights Reserved.

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

Bent Borg Jensen and Raymond P. Cox

Institute of Biochemistry, Odense University, DK-5230 Odense M, Denmark

ABSTRACT

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 K_m for N₂ of 65 µM at 35°C, corresponding to 0.121 atmosphere of N₂. 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 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.

Present address: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706.