The flavodoxins from Azotobacter vinelandii cells grown N2-fixing and from cells grown on NH40Ac have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I,11, 111) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M KC1, Fld I1 at 0.43 M KC1 and Fld I11 at 0.45 M KC1. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25 "C) of the semiquinone/ hydroquinone couple, which was -320 mV for Fld I and -500 mV for the other two flavodoxins (Fld 11 and Fld 111).All three flavodoxins were present in cells grown on NH40Ac. In cells grown on N2 as N source only Fld 1. and Fld I1 were found. The concentration of Fld I1 was 10-fold higher in N2-fixing cells than in cells grown on NH40Ac. Evidence has been obtained that Fld I1 is involved in electron transport to nitrogenase.As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.Flavodoxin from Azotobacter vinelandii was first isolated by Shethna et al. in 1964 [l, The protein consists of a single polypeptide chain with 179 amino acid residues, it contains one FMN and has a relative molecular mass of 19990 [S]. There is one single cysteine residue present which can cause dimerization of two flavodoxin molecules, a process which results in the loss of biological activity [9]. In addition to the 5'-phosphate ester on the FMN, flavodoxin contains 2 mol tightly bound phosphate/mol [lo]. One phosphate group is covalently bound to the protein in a phosphodiester linkage between serine and threonine residues. It has been suggested that the other is an acid-labile phosphate in an acyl phosphate linkage with a protein COOH group [ll]. At pH 8.0 and 25°C the redox potential of the quinone/semiquinone couple (E2) of flavodoxin is -250 mV [12-141. However an anomalous value of + 50 mV was also reported for E2 [15]. The redox potential of the semiquinone/hydroquinone couple ( E l ) is -500 mV The primary function of the flavodoxin in Azotobacter species was suggested to be electron transport to nitrogenase. [12-161.In 1969, Benemann et al. [4, 171 showed that flavodoxin was one of the four factors native to A. vinelandii cells needed for electron transport from NADPH to nitrogenase; however, the reported rate was just a fraction of the activity obtained with dithionite as electron donor. It appeared that the endogenous enzyme system was not capable of reducing flavodoxin effectively beyond the semiquinone state, whereas the hydroquinone form is necessary for nitrogenase activity [18, 191. In fact, completely reduced flavodoxin was found to be a good electron donor for nitrogenase, activities being 50% higher than with dithionite [I9 -211. Furthermore flavodoxin from Azotobacter chroococcurn was shown to be nif specific [=I.What argues against flavodoxin being the unique physiological electron donor for ni...

Nodule‐specific root proteins – so called nodulins – were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nod+fix‐ and a pop ‐fix‐) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.

The influence of the growth conditions on the concentration of nitrogenase and on the nitrogenase activity, was studied in intact Azotobacter vinelandii cells. It was observed that whole cell nitrogenase activity could be enhanced in two ways. An increase of the growth rate of cell was accompanied by an increase in whole cell nitogenase activity and by an increase in the concentration of nitrogenase in the cells. The molar ratio of Fe protein: MoFe protein was 1.47 ± 0.17 and independent of the growth rate. Activity measurements in cell extracts showed that the catalytic activity of the nitrogenase proteins was independent of the growth rate of cells. The second way to increase whole cell nitrogenase activity was to expose cells to excess oxygen. Whole cells were exposed for 2.5 h to an enhanced oxygen‐input rate. After this incubation nitrogenase activity was increased without an increase in protein concentration. It is calculated that the catalytic activity of the Fe protein in these cells was 6200 nmol C2H4 formed · min−1. With these cells and with cells grown at a high growth rate, 50% of the whole cell activity is lost by preparing a cell‐free extract. It will be demonstrated that this inactivation is partly caused by the activity measurements in vitro. When dithionite was replaced by flavodoxin as electron donor, a maximal catalytic activity of 4500 nmol C2H4 formed · min −1· (mg Fe protein)−1 was measured in vitro for the Fe protein. The results are discussed in relation to the present model for nitrogenase catalysis

The involvement of the cytoplasmic membrane in electron transport to nitrogenase has been studied. Evidence shows that nitrogenase activity in Azotobacter vinelandii is coupled to the flux of electrons through the respiratory chain.To obtain information about proteins involved, the changes occurring in A. vinelandii cells transferred to nitrogen-free medium after growth on NH4CI (derepression of nitrogenase activity) were studied. Synthesis of the nitrogenase polypeptides was detectable 5 min after transfer to nitrogen-free medium. No nitrogenase activity could be detected until t = 20 min, whereupon a linear increase of nitrogenase activity with time was observed. Synthesis of nitrogenase was accompanied by synthesis of flavodoxin I1 and two membrane-bound polypeptides of M , 29000 and 30000. Analysis with respect to changes in membrane-bound NAD(P)H dehydrogenase activities revealed the induction of an NADPH dehydrogenase activity, which was not detectable in membranes isolated from cells grown in the presence of NH40Ac. This induced activity was associated with the appearance of a polypeptide of M , 29 000 in the NADPH dehydrogenase complex.The enzyme nitrogenase is capable of reducing atmospheric N2 to ammonia. For activity the enzyme needs an anaerobic environment, MgATP and a strong reductant. How this reductant is generated in the aerobic nitrogen-fixing bacterium Azotobacter vinelandii is not well understood. In 1971 Benemann et al.[l] proposed a linear electron transfer pathway from NADPH to nitrogenase, including both ferredoxin and flavodoxin. However the nitrogenase activity measured with endogenous proteins was less than 5% of the activity with dithionite as electron donor. Haaker et al. [2, 31 have critisized the proposed model as being too simplistic with respect to the redox potentials of the different components of the electron transfer chain. It has been shown by the same authors [4] that a high proton motive force across the cytoplasmic membrane is required for nitrogenase activity. The membrane potential was found to be an especially important factor [5]. Recently it has been shown that A . vinelandii is able to make at least three different flavodoxins [6]. Flavodoxin I1 is most likely to be the reductant for nitrogenase [6]. Whether ferredoxin is involved in electron transport to nitrogenase in vivo is uncertain [6, 71. An enzyme system capable of reducing flavodoxin I1 has still to be found. This paper describes the changes observed in A . vinelandii cells transferred to nitrogen-free medium after growth in the presence of NH4Cl. For Azotobacter species it is known that, when sufficient NH; is supplied to cells, synthesis of the nitrogenase proteins [S] and possibly also 13 other nij-specific polypeptides [9] is repressed. It has been investigated whether, during derepression, the (membrane) proteins necessary for electron transport to nitrogenase are synthesized simultaneously with the nitrogenase proteins. In addition, a comparison has been made between the membrane-bound NAD(P)H dehy...

In Azotobacter vinelandii cells, the short-term inhibition of nitrogenase activity by NH4Cl was found to depend on several factors. The first factor is the dissolved oxygen concentration during the assay of nitrogenase. When cells are incubated with low concentrations of oxygen, nitrogenase activity is low and ammonia inhibits strongly. With more oxygen, nitrogenase activity increases. Cells incubated with an optimum amount of oxygen have maximum nitrogenase activity, and the extent of inhibition by ammonia is small. With higher amounts of oxygen, the nitrogenase activity of the cells is decreased and strongly inhibited by ammonia. The second factor found to be important for the inhibition of nitrogenase activity by NH4Cl was the pH of the medium. At a low pH, NH4' inhibits more strongly than at a higher pH. The third factor that influenced the extent of ammonia inhibition was the respiration rate of the cells. When cells are grown with excess oxygen, the respiration rate of the cells is high and inhibition of nitrogenase activity by ammonia is small. Cells grown under oxygen-limited conditions have a low respiration rate and NH4Cl inhibition of nitrogenase activity is strong. Our results explain the contradictory reports descnrbed in the literature for the NH4Cl inhibition of nitrogenase in A. vinelandii.