FeMo cofactor biosynthesis in a nifE(-) mutant of Rhodobacter capsulatus

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 purpl e bacterium Rhodobacter capsulatus with mutations in nifE, as well as in th e operon harbouring the nifE gene, were capable of reducing acetylene and g rowing diazotrophically, although at distinctly lower rates than the wild-t ype strain. The diminished rates of substrate reduction were found to corre late with the decreased amounts of the dinitrogenase component (MoFe protei n) expressed in R. capsulatus. The in vivo activity, as measured by the rou tine 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 muM) Moreover, in these strains a high proportion of ethane was produced from acetylene (appr oximate to 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production wa s always found to be less than 1%. The isolation and partial purification o f the MoFe protein from the nifE mutant strain by Q-Sepharose chromatograph y and subsequent analysis by EPR spectroscopy and inductively coupled plasm a MS revealed that FeMoco is actually incorporated into the protein (1.7 mo lecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosyn thetic pathway of the FeMoco demands reconsideration. It is shown for the f irst time that NifNE is not essential for biosynthesis of the cofactor, alt hough its presence guarantees formation of a higher content of intact FeMoc o-containing MoFe protein molecules. The implications of our findings for t he biosynthesis of the FeMoco will be discussed.