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.

Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus

“…The absence or the almost complete absence of Mo in the growth medium is certainly the most fundamental prerequisite for the derepression of the Moindependent Fe nitrogenase in R. cupsulatus [ 10,21,241. However, there are several further growth factors that also significantly influence the expression of Fe nitrogenase activity.…”

Metalloclusters of the nitrogenases

“…The ability of such deletion strains to grow on N2 under the appropriate conditions of Mo deprivation, V availability and the absence of Mo and V indicated that Mo-independent routes for N2 fixation existed [5]. A similar strategy has recently been used to show that the photosynthetic diazotroph Rhodobacter capsulatus has the third nitrogenase in addition to Mo nitrogenase [6].…”

“…The ability of such deletion strains to grow on N2 under the appropriate conditions of Mo deprivation, V availability and the absence of Mo and V indicated that Mo-independent routes for N2 fixation existed [5]. A similar strategy has recently been used to show that the photosynthetic diazotroph Rhodobacter capsulatus has the third nitrogenase in addition to Mo nitrogenase [6].In addition to those organisms from which Mo-independent nitrogenases have been isolated, there are physiological and genetic data indicating a wider distribution of these systems [7]. The stimulation of N2-dependent growth by V added to medium depleted of metals, and the reduction of CzHz to form some CzH6 (a characteristic of Mo-independent nitrogenases) are observed with the cyanobacterium Anahaena variabilis, the anaerobe Clostridium pasteurianum and the archaebacterium Methanosarcina barkeri.…”

Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum

“…Recently, two other diazotrophs, Rhodobacter capsulatus (16,20) and Rhodospirillum rubrum (12,13), have been found to have alternative nitrogenases that are similar to nitrogenase 3 from A. vinelandii. Since Mo-independent nitrogenases are likely to be found in a wide range of diazotrophs, the existence of these enzymes poses interesting questions concerning the role(s) that they might play under natural conditions.…”