FeMo cofactor biosynthesis in a nifE⁻ mutant of Rhodobacter capsulatus

Abstract

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20±50 mm). Moreover, in these strains a high proportion of ethane was produced from acetylene (< 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer).On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.Keywords: acetylene reduction; EPR spectroscopy; MoFe protein; nitrogen fixation; nitrogenase.The biological reduction of molecular nitrogen (N 2 ) is one of nature's most fundamental processes, providing nitrogen for plant growth. This process, which is mediated by the enzyme nitrogenase, has been found to take place exclusively in a small number of bacteria and archaea. Three closely related, but genetically distinct types of nitrogenase system (nif, vnf, anf) have been shown to occur in nitrogen-fixing organisms (reviewed in [1]). The most common and best characterized system is the conventional molybdenum-containing nitrogenase (nif) that is present in all diazotrophs. The two alternative nitrogenase systems have been proven to be Mo-independent, one containing vanadium (vnf), the other lacking both molybdenum and vanadium (Fe-only nitrogenase, anf). All nitrogenases are composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase. A further but completely different alternative nitrogenase present in Streptomyces thermoautotrophicus has also been described [2]. This type of ni...

Accumulation of 99Mo-containing Iron-Molybdenum Cofactor Precursors of Nitrogenase on NifNE, NifH, and NifX ofAzotobacter vinelandii

“…In A. vinelandii, NifNE is absolutely required for the biosynthesis of FeMo-co as nifN or E mutants show a definite nif Ϫ phenotype. However, a recent report by Siemann et al (31) reveals that in Rhodobacter capsulatus NifNE is not essential for the biosynthesis of FeMo-co, though its presence greatly enhances the content of intact FeMo-co-containing NifDK.…”

Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii

“…It is noteworthy that no additional proteins are needed for the interaction between NafY and NifB-co. If the binding of NafY to NifB-co is relevant under physiological conditions, it may have some implications in our understanding of the biosynthesis of FeMo-co. Siemann et al (35) describe a residual FeMo-co biosynthetic activity in a nifE mutant strain of Rhodobacter capsulatus. The authors suggest that, in the absence of NifNE, NifB-co could be further transformed into FeMo-co while bound to a NafY-like protein.…”

Nitrogen Fixation in the Photosynthetic Purple Bacterium Rhodobacter capsulatus

“…R. capsulatus nifE and nifN mutants exhibit a Nif phenotype but can grow diazotrophically via the alternative nitrogenase . In contrast to other diazotrophic bacteria, the nifE gene product is not absolutely essential for biosynthesis of FeMoco, although the presence of NifE/NifN guarantees formation of a much higher intracellular concentration of MoFe proteins containing FeMoco (Siemann et al, 2001). Despite the requirement of NifX for in vitro biosynthesis of FeMoco in A. vinelandii (Rangaraj et al, 2001;Shah et al, 1999), nifX mutations in A. vinelandii, K. pneumoniae or R. capsulatus have little effect on nitrogenase activity (Gosink et al, 1990;Jacobson et al, 1989;Moreno-Vivian et al, 1989b).…”