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 > 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 (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. Copyright © 1991, Wiley Blackwell. All rights reserved