The Flavodoxin Reduced All-Ferrous Fe Protein in Nitrogenase

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Tom Lowery Jr. and Dr. Gerald D. Watt, Chemistry and Biochemistry

Nitrogenase, an enzyme system found in a small, diverse group of diazotrophic microorganisms, is responsible for the natural reduction of atmospheric N2 to NH3. Nitrogenase plays a critical role in the earth’s nitrogen cycle and has therefore been a subject of intense study for over thirty five years. Nevertheless, detailed mechanistic explanations of in vivo nitrogenase catalysis remain ambiguous.1

Nitrogenase consists of two proteins: the iron protein and molybdenum-iron protein, Fe and MoFe proteins, respectively. The Fe protein contains a redox-active [Fe4S4] metal cluster, which is reduced by an in vivo or in vitro reductant. Reduced Fe protein bound to two MgATPs can then transfer its excess electron(s) to the MoFe protein, with concomitant hydrolysis of the ATP.1 Electrons accumulate on the MoFe protein’s metal cofactor, where substrates (N2 and H+) are processed.

Until recently, the Fe protein was thought only to operate in the [Fe4S4]2+/[Fe4S4]1+ redox couple that transfers a single electron to the MoFe protein per Fe-MoFe protein interaction as characterized under the non-physiological reductant, sodium hydrosulfite.1 By using an alternative non-physiological reductant, titanium III citrate, a new Fe protein oxidation state, [Fe4S4]0 (all-ferrous), was recently discovered.2-4 The all-ferrous Fe protein has been shown to participate in the [Fe4S4]2+/[Fe4S4]0 redox couple, transferring two electrons per Fe-MoFe protein interaction. The understanding of nitrogenase has been complicated by this discovery because the [Fe4S4]2+/[Fe4S4]0 redox couple cuts in half the required amount of ATP hydrolyzed for a given amount of product formed during nitrogenase catalysis.1−3 As ATP utilization is critical for physiological pathways, determining which redox couple operates using the in vivo reductant, flavodoxin (FlpH2), has become an important concern.1−4 Previous researchers who reduced the Fe protein with FlpH2 observed that FlpH2 can form the all-ferrous Fe protein, which is inconsistent with the conventional assumption that flavodoxin can only form the [Fe4S4]1+ Fe protein oxidation state.4

The present study presents further evidence that excess FlpH2 indeed forms the all-ferrous Fe protein. All proteins were isolated and purified from Azotobacter vinelandii (Av) as described elsewhere.5 The results from spectrometric titrations of FlpH2 with different oxidation states of the Fe protein, and preliminary qualitative EPR analyses of the FlpH2 reduced Fe protein are presented below. Also, an adaptation of the Evans NMR method developed to measure the paramagnetic susceptibility of the Fe protein metal cluster under different reductants is discussed below.6 Quantitative EPR analyses and the paramagnetic susceptibility of the FlpH2 reduced allferrous Fe protein were not completed because stock Fe protein quantities were depleted. These experiments will be completed after sufficient amounts of Av Fe protein have been isolated and purified.

Spectrometric Titration. The Fe protein oxidation state formed by FlpH2 was determined by spectrometric titration conducted on a Hewlett Packard 8453 UV Spectrometer in a glove box at oxygen levels below 1 ppm. A quantitative amount of purified Fe protein was added to excess FlpH2 in a buffered solution of 50 mM Tris pH 8.0 buffer. As FlpH2 transfers electrons to the Fe protein, the formation of the dark blue oxidized flavodoxin (Flp· ) was measured by the solution’s increased absorbency at 580 nm. The ratio between the moles of Flp· formed and the moles of Fe protein added was used to calculate the number of electrons transferred per Fe protein.4 Multiple experiments showed that no electrons are transferred from excess FlpH2 to the [Fe4S4]0 oxidation state of the Fe protein, 0.9 ± 0.1 electrons are transferred to [Fe4S4]1+, and 2.0 ± 0.2 electrons are transferred to [Fe4S4]2+. A control was also conducted by adding K3Fe(CN)6 to excess FlpH2, which showed that 1.0 ± 0.1 electrons are transferred from excess FlpH2 to K3Fe(CN)6. These results indicate that excess FlpH2 forms the [Fe4S4]0 state of the Fe protein.

Spectroscopic comparison. Spectroscopic comparisons of different Fe protein oxidation states using electron paramagnetic resonance (EPR) were possible because the different Fe protein oxidation states have unique EPR spectra.3,7 Because FlpH2 and the Fe protein are similar in size and charge, separation of the two has proved difficult. Therefore, EPR measurements of the FlpH2 reduced Fe protein were made in the presence of excess FlpH2 and Flp· . The EPR signal from Flp· was subtracted from the FlpH2, Flp· , Fe protein sample by measuring a control sample made at the same FlpH2, Flp· concentrations and at K3Fe(CN)6 concentrations equal to the [Fe4S4]1+ Fe protein concentration in the FlpH2, Flp· , Fe protein samples. Preliminary qualitative results indicate that the all-ferrous state of the Fe protein formed by FlpH2 may be different than the titanium III citrate all-ferrous Fe protein by having a spin = 0 metal cluster instead of a spin = 4 metal cluster.

NMR Magnetic Susceptibility of [Fe4S4]0, [Fe4S4]1+, [Fe4S4]2+. The Evans NMR method was adapted to determine the magnetic susceptibility of each oxidation state of the Fe protein.6 The reference and sample solutions were 7% t-butyl alcohol and 0.6% t-butyl alcohol, respectively. The balance of both solutions was 20% D2O, 80% 50 mM Tris pH 7.4 buffer. Controls were run using varying concentrations of MnSO4 and CuSO4. All Fe protein samples were prepared in anaerobic buffers and separated from reductant on anaerobic P-2 Biogel columns in the glove box. All NMR measurements were taken on a Varian 500 MHz or INOVA 300 MHz nuclear magnetic resonance spectrometer. I have measured the paramagnetic susceptibility of the sodium hydrosulfite reduced [Fe4S4]1+ and titanium III citrate reduced [Fe4S4]0 Fe proteins using the diamagnetic methylene blue oxidized [Fe4S4]2+ Fe protein as a control. In the future, I plan to measure the magnetic susceptibility of the FlpH2-reduced, all-ferrous Fe protein to determine it unique characteristics.8

References

  1. Rees, D. C., Howard, J. B. (2000) Curr. Opin. Chem. Biol. 4, 559–566.
  2. Erickson, A. J., Nyborg, A. C., Johnson, J. L., Truscott, S. M., Gunn, A., Nordmeyer, F. R., Watt, G. D. (1999) Biochemistry 38, 14279–14285.
  3. Angove, H. C., Sun, J. Y., Burgess, B. K., Münck, E. (1997) J. Am. Chem. Soc. 119, 8730–8731.
  4. Thiriot, D. (1995) M.S. Thesis. Brigham Young University, Provo, UT.
  5. Burgess, B. K., Jacobs, D. B., Stiefel, E. I. (1980) Biochim. Biophys. Acta 614, 196−209.
  6. Evans, D. F. (1959) J. Chem. Soc. 2003−2005.
  7. Lindahl, P. A., Edmund, P. D., Thomas, A. K., William, H. O, Muenck E. M. (1985) J. Biol. Chem. 260, 11160−11173.
  8. I thank Dr. Richard Watt at the University of New Mexico for analyzing the EPR samples and Dr. Roger G. Harrison for assisting me with NMR sample analysis. This work was partially supported by a Brigham Young University ORCA competitive undergraduate fellowship and by Brigham Young University Department of Chemistry and Biochemistry.