The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis.

Abstract

The requirement for both an electron donor and ATP for the enzymatic reduction of N2 to ammonia was demonstrated with extracts of Clostridium pasteurianum,1-4 Azotobacter vinelandii,5 7 and Rhodospirillum rubrum.' 8 With hydrosulfite as the electron donor and an ATP-generating system (creatine phosphokinase), N2-reducing preparations from A. vinelandii and R. rubrum were shown to catalyze a concomitant ATP-dependent H2-evolution reaction.6-9 This reaction was subsequently demonstrated with extracts of C. pasteurianum10' 11 by inhibiting the classical hydrogenase with carbon monoxide. The use of hydrosulfite as the electron donor, in combination with an ATP-generating system, provides a useful assay reaction for enzyme purification since both required components can be added exogenously. We reported previously7 that the N2-reducing and ATP-dependent H2-evolving activities could be obtained from azotobacter extracts by precipitation with protamine followed by pH fractionation, and that these fractions contained a high level of nonheme iron as well as molybdenum. Procedures have now been developed for the isolation of two enzyme fractions, both of which are required for N2 reduction, ATP-dependent H2 evolution, and the related release of inorganic phosphate. The first enzyme contains both nonheme iron and molybdenum; the second contains nonheme iron but no molybdenum. The purification and some properties of these two enzymes are described in this report. A preliminary report was presented previously.12 Mlortenson'3 14 recently reported evidence suggesting the requirement for at least two enzymes from C. pasteurianum extracts for the catalysis of N2 reduction and related reactions. Materials and Methods.-Azotobacter vinelandii 0 was cultured and harvested as previously described,6 and the cell paste either used when harvested or stored frozen in an argon atmosphere. Cells (100-120-gm cell paste) were ruptured in a French pressure cell, and the S1441/2 supernatant

Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens

“…However, genetic studies indicate that neither NrfA nor NrfH is required for Fe(III) reduction by G. sulfurreducens (Table 5). It should also be considered that this increased expression of nrfA and nrfH may be related to nutrient limitation associated with the Fe(III) oxide medium, as increased expression of NrfA and NrfH has been associated with nitrogen limitation (Bulen & LeComte, 1966;Hageman & Burris, 1978).…”

Transcriptome of Geobacter uraniireducens growing in uranium-contaminated subsurface sediments

“…Although increased expression of proteins involved in nitrogen fixation is frequently associated with nitrogen limitation (Bulen and LeComte, 1966;Hageman and Burris, 1978), studies have also shown that under conditions of nitrogen limitation, nitrite reductases can be involved in the anabolic incorporation of nitrogen into biomolecules Nakano et al, 1998). Therefore, it is possible that the increased expression of the nitrite reductase genes, nrfA and nrfH, in sedimentgrown cells is the result of nitrogen limitation.…”

Regulation of Nitrogen Fixation by Fe‐S Protein II in Azotobacter vinelandii

“…In 1966 Bulen and Le Comte [30] found in their purified nitrogenase preparations ((242-1) a pinkish protein fraction, that could be separated from the nitrogenase proteins by DEAE-cellulose chromatography. The fraction could be eluted from the column with 0.15 M NaCI.…”