Characterization of three different flavodoxins from Azotobacter vinelandii

Abstract

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...

Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus

“…Their oxidized absorption spectra show significant differences in the position of long-wave-length maxima (450 nm for R. capslulatlus and 460 nm for R. rubrum) which may reflect a slightly different arrangement of flavin mononucleotide in the protein. The analogous maxima for the nifspecific flavodoxins from K pneumoniae and A. vinelandii are found at 452 to 454 nm (7,20,25 (17). Thus, the flavodoxin isolated here is a real candidate for the physiological reductant of nitrogenase in R. capsulatus.…”

Last In, First Out

“…The holoflavodoxin concentration was determined spectrophotometrically using an extinction coefficient of 11.3 mM Ϫ1 cm Ϫ1 at 452 nm (15). Apoflavodoxin was subsequently prepared by trichloroacetic acid precipitation (11, 16) followed by gel filtration on a Superdex 200 prep grade column (Amersham Biosciences) to remove apoflavodoxin molecules in an oligomeric state (13).…”

Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii

“…These gels give a better resolution but for unknown reasons the relative molecular mass of flavodoxin is slightly higher compared with the regular Laemmli gels. For molecular mass markers see the preceding paper [6]. Nitrogenase activity of whole cells was measured at different oxygen input rates as described earlier [15].…”

“…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].…”

“…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.…”