Detection of the in vivo incorporation of a metal cluster into a protein

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Abstract
The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo‐independent nitrogen‐fixing system, an ‘iron‐only’ nitrogenase which is strongly repressed by molybdate. The MoO2− concentration causing 50% repression of the alternative nitrogenase in nifHDK− cells was 6 nM. If MoO42− was added to a growing nifHDK− culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H6 formation decreased continously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yeild of C2H6 increased from 2% (C2H6/C2H4X100) in the absence of MoO42−, to a maximal value of 69% in the presence of MoO42− (1 mM) after 72 h incubation. This ‘Mo effect’ appeared to be stronger the higher the MoO42− concentration in the medium and the longer the incubation time. In the presence of ReO4−, WO42− or VO43−, a similar effect did not occur. The ‘Mo effect’ was not observed in a nifHDK−nifE− double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK−nifQ− mutant. Crude extracts from nifHDK− cells cultivated in the presence of MoO42−, also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S= 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the ‘Mo effect’ is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase. The ‘Mo effect’ was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the ‘Mo effect’ appeared, therefore, to be independent of de‐novo protein synthesis. The analysis of nifE‐lacZ and nifN‐lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO42− deficiency. The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.