DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS

Schneider K, Müller A, Schramm U, Klipp W (1991)
EUROPEAN JOURNAL OF BIOCHEMISTRY 195(3): 653-661.

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Abstract
In Rhodobacter capsulatus there exists, in addition to a conventional Mo-containing nitrogenase, a second, Mo-independent nitrogenase which was demonstrated in wild-type cell as well as in cells of a nifHDK- mutant. To construct this R. capsulatus mutant, a 4-kb Bg/II-HindIII fragment encompassing nifK, nifD and most of the nifH coding region was substituted by an interposon coding for kanamycin resistance. The alternative nitrogenase is repressed by molybdenum. Mo concentration > 1 ppb in the growth medium prevented diazotrophic growth of nifHDK- cells and the expression of nitrogenase activity. The Mo-independent nitrogenase was maximally derepressed in activated carbon-treated media which contained < 0.05 ppb Mo, high concentrations of iron (1 mM ferric citrate) and serine as N source. Under N2-fixing and optimal Mo-deficient conditions, nifHDK- cells grew with a doubling time of 9 h. The highest activity achieved with whole cells was 1.2 nmol ethylene . min-1).mg protein-1. Vanadium neither stimulated nor inhibited growth and activity. The alternative nitrogenase reduced acetylene to both ethylene and ethane. With whole cells (nifHDK-) the proportion of ethane varied over 2-5% depending on the amount of residual traces of Mo in the medium. The addition of Mo to a growing, nitrogenase-active culture resulted in a slow decrease of total activity but also in a simultaneous increase of ethane production up to 40%. In contrast, cell-free extracts and the purified enzyme did not show any or only very little ethane formation (0-0.4%). Both enzyme components appeared to be very labile proteins. Component 2 lost almost all its activity during cell breakage. With component 1 in crude extracts, if complemented with the stable component 2 of the Mo-nitrogenase from Xanthobacter autotrophicus, a recovery of 50% of the original whole cell activity could be achieved. During purification, component 1 (from the nifHDK-mutant) remained remarkably stable. The partially purified component 1 had a pH optimum (acetylene reduction) of 7.8-8.0, relatively high affinity to acetylene (K(m) = 0.055 mM) and was analyzed to contain 20 mol Fe atoms/mol protein, 0.2 mol Mo atoms and negligible amounts of V, W and Re. The dithionite-reduced dinitrogenase appeared to be ESR-silent. The results indicate that the alternative nitrogenase of R. capsulatus is not a vanadium enzyme but rather a heterometal-free Fe-nitrogenase or a nitrogenase with an as-yet-unidentified heterometal atom.
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Schneider K, Müller A, Schramm U, Klipp W. DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS. EUROPEAN JOURNAL OF BIOCHEMISTRY. 1991;195(3):653-661.
Schneider, K., Müller, A., Schramm, U., & Klipp, W. (1991). DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS. EUROPEAN JOURNAL OF BIOCHEMISTRY, 195(3), 653-661.
Schneider, K., Müller, A., Schramm, U., and Klipp, W. (1991). DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS. EUROPEAN JOURNAL OF BIOCHEMISTRY 195, 653-661.
Schneider, K., et al., 1991. DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS. EUROPEAN JOURNAL OF BIOCHEMISTRY, 195(3), p 653-661.
K. Schneider, et al., “DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS”, EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 195, 1991, pp. 653-661.
Schneider, K., Müller, A., Schramm, U., Klipp, W.: DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS. EUROPEAN JOURNAL OF BIOCHEMISTRY. 195, 653-661 (1991).
Schneider, Klaus, Müller, Achim, Schramm, U, and Klipp, W. “DEMONSTRATION OF A MOLYBDENUM-INDEPENDENT AND VANADIUM-INDEPENDENT NITROGENASE IN A NIFHDK-DELETION MUTANT OF RHODOBACTER-CAPSULATUS”. EUROPEAN JOURNAL OF BIOCHEMISTRY 195.3 (1991): 653-661.
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