Demonstration of a molybdenum- and vanadium-independent nitrogenase in a nifHDK-deletion mutant of Rhodobacter capsulatus

Abstract

In Rhodobacter capsulatus there exists, in addition to a conventional Mo-containing nitrogenase, a second, Mo-indendent nitrogenase which was demonstrated in wild-type cells as well as in cells of a nifHDK- mutant. To construct this R. capsulatus mutant, a 4-kb BglII-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 greater than 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 less than 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 (Km = 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.