Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus

Siemann S, Schneider K, Oley M, Müller A (2003)
BIOCHEMISTRY 42(13): 3846-3857.

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Abstract
In the phototrophic non-sulfur bacterium Rhodobacter capsulatus, the biosynthesis of the conventional Mo-nitrogenase is strictly Mo-regulated. Significant amounts of both dinitrogenase and dinitrogenase reductase were only formed when the growth medium was supplemented with molybdate (I muM). During cell growth under Mo-deficient conditions, tungstate, at high concentrations (I mM), was capable of partially (similar to25%) substituting for molybdate in the induction of nitrogenase synthesis. On the basis of such conditions, a tungsten-substituted nitrogenase was isolated from R. capsulatus with the aid of anfA (Fe-only nitrogenase defective) mutant cells and partially purified by Q-sepharose chromatography. Metal analyses revealed the protein to contain an average of I W-, 16 Fe-, and less than 0.01 Mo atoms per alpha(2)beta(2)-tetramer. The tungsten-substituted (WFe) protein was inactive in reducing N-2 and marginally active in acetylene reduction, but it was found to show considerable activity with respect to the generation of H-2 from protons. The EPR spectrum of the WFe protein, recorded at 4 K, exhibited three distinct signals: (i) an S = 3/2 signal, which dominates the low-field region of the spectrum (g = 4.19, 3.93) and is indicative of a tungsten-substituted cofactor (termed FeWco), (ii) a marginal S = 3/2 signal (g = 4.29, 3.67) that can be attributed to residual amounts of FeMoco present in the protein, and (iii) a broad S = 1/2 signal (g = 2.09, 1.95, 1.86) arising from at least two paramagnetic species. Redox titrational analysis of the WFe protein revealed the midpoint potential of the FeWco (E-m < -200 mV) to be shifted to distinctly lower potentials as compared to that of the FeMoco (E-m similar to -50 mV) present in the native enzyme. The P clusters of both the WFe and the MoFe protein appear indistinguishable with respect to their midpoint potentials. EPR spectra recorded with the WFe protein under turnover conditions exhibited a 20% decrease in the intensity of the FeWco signal, indicating that the cofactor can be enzymatically reduced only to a small extent. The data presented in the current study demonstrate the pivotal role of molybdenum in optimal N-2 fixation and provides direct evidence that the inability of a tungsten-substituted nitrogenase to reduce N-2 is due to the difficulty to effectively reduce the FeW cofactor beyond its semireduced state.
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Siemann S, Schneider K, Oley M, Müller A. Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus. BIOCHEMISTRY. 2003;42(13):3846-3857.
Siemann, S., Schneider, K., Oley, M., & Müller, A. (2003). Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus. BIOCHEMISTRY, 42(13), 3846-3857.
Siemann, S., Schneider, K., Oley, M., and Müller, A. (2003). Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus. BIOCHEMISTRY 42, 3846-3857.
Siemann, S., et al., 2003. Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus. BIOCHEMISTRY, 42(13), p 3846-3857.
S. Siemann, et al., “Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus”, BIOCHEMISTRY, vol. 42, 2003, pp. 3846-3857.
Siemann, S., Schneider, K., Oley, M., Müller, A.: Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus. BIOCHEMISTRY. 42, 3846-3857 (2003).
Siemann, S, Schneider, Klaus, Oley, M, and Müller, Achim. “Characterization of a tungsten-substituted nitrogenase isolated from Rhodobacter capsulatus”. BIOCHEMISTRY 42.13 (2003): 3846-3857.
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