Estimation of nitrogenase activity in the presence of ethylene biosynthesis by use of deuterated acetylene as a substrate

• Published in: Applied and Environmental Microbiology Vol. 55; no. 2; pp. 354 - 359
• Main Authors: Lin-Vien, D, Fateley, W.G, Davis, L.C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.02.1989
• Subjects: 550501 - Metabolism- Tracer Techniques; 560300 - Chemicals Metabolism & Toxicology; ACETILENO; ACETYLENE; Acetylene - metabolism; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALKENES; ALKYNES; BACTERIA; Bacteriological methods and techniques used in bacteriology; Bacteriology; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; BIOSYNTHESIS; Bradyrhizobium japonicum; CHROMATOGRAPHY; DEUTERIUM; ENZYME ACTIVITY; ENZYMES; ETHYLENE; Ethylenes - metabolism; ETILENO; FOOD; FOURIER TRANSFORMATION; Fundamental and applied biological sciences. Psychology; GAS CHROMATOGRAPHY; GLYCINE MAX; HYDROCARBONS; HYDROGEN ISOTOPES; INTEGRAL TRANSFORMATIONS; ISOTOPE APPLICATIONS; ISOTOPES; KLEBSIELLA; Klebsiella pneumoniae; Klebsiella pneumoniae - enzymology; Klebsiella pneumoniae - genetics; LIGHT NUCLEI; METABOLISM; Microbiology; MICROORGANISMS; Mutation; NITRO-GROUP DEHYDROGENASES; NITROGENASA; NITROGENASE; Nitrogenase - metabolism; NUCLEI; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; OXIDOREDUCTASES; PLANTS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; REDUCCION; REDUCTION; RHIZOBIUM; Rhizobium - enzymology; SEPARATION PROCESSES; SOYBEANS; SPECTROPHOTOMETRY; Spectrophotometry, Infrared - methods; STABLE ISOTOPES; STOICHIOMETRY; SYNTHESIS; TRACER TECHNIQUES; TRANSFORMATIONS; VEGETABLES; Deuterium; Vegetals; Isotope labelling; Enzyme; Coupled method; Bradyrhizobium japonicum; Ethylene; Fourier spectrometry; Acetylene; Infrared spectrometry; Substrate; Gas chromatography; Enzymatic activity; Nitrogenase; Nitrogen fixation; Bacteria; Rhizobiaceae; Klebsielleae; Klebsiella pneumoniae; Enterobacteriaceae; Quantitative analysis
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.55.2.354-359.1989

Nitrogenase reduces deuterated acetylene primarily to cis dideuterated ethylene. This can be distinguished from undeuterated ethylene by the use of Fourier transform infrared spectroscopy. Characteristic bands in the region from 800 to 3,500 cm-1 can be used to identify and quantitate levels of these products. This technique is applicable to field studies of nitrogen fixation where ethylene biosynthesis by plants or bacteria is occuring. We have verified the reaction stoichiometry by using Klebsiella pneumoniae and Bradyrhizobium japonicum in soybeans. The most useful bands for quantitation of substrate purity and product distribution are as follows: acetylene-do, 3,374 cm-1; acetylene-d1, 2,584 cm-1; acetylene-d2, 2439 cm-1; cis-ethylene-d2, 843 cm-1; trans-ethylene-d2, 988 cm-1; ethylene-d1, 943 cm-1; ethylene-do, 949 cm-1. (The various deuterated ethylenes and acetylenes are designated by a lowercase d and subscript to indicate the number, but not the position, of deuterium atoms in the molecule.) Mass spectrometry coupled to a gas chromatograph system has been used to assist in quantitation of the substrate and product distributions. Significant amounts of trans-ethylene-d2 were produced by both wild-type and nifV mutant K. pneumoniae. Less of this product was observed with the soybean system