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Identification of molybdoproteins in Clostridium pasteurianum
Abstract:
Cells of Clostridium pasteurianum whose N source is switched from NH3 to N2 accumulate large amounts of molybdenum beginning 1.5 h before the detection of nitrogenase activity. Anaerobic multiphasic gel electrophoresis and anion-exchange chromatography were used to identify the molybdoproteins and molybdenum-containing components present in N2-fixing cells. In addition to molybdate, six distinct 99Mo-labeled species were detected, i.e., a membrane fragment, the MoFe protein of nitrogenase, formate dehydrogenase, a Mo "binding-storage" protein, a 30-kilodalton molybdoprotein, and a low-molecular-weight molybdenum species. Of these, the MoFe protein, formate dehydrogenase, and the Mo binding-storage protein were present in more than one zone because of complex formation with other proteins, partial denaturation, and variation in the amount of Mo bound to the protein, respectively. In addition to the six proteins, a soluble "free" Mo cofactor in the cytosol was detected by showing that it reconstituted nitrate reductase activity in crude extracts of the Neurospora crassa mutant nit-1.
Insights
Clostridium pasteurianum cells accumulate molybdenum before nitrogenase activity, identifying key molybdoproteins like the MoFe protein and a novel Mo cofactor crucial for nitrogen fixation.
Area of Science:
- Biochemistry
- Microbiology
- Molecular Biology
Background:
- Nitrogen fixation is a vital microbial process.
- Molybdenum is an essential trace element for nitrogenase activity.
- Understanding molybdenum metabolism in nitrogen-fixing bacteria is crucial.
Purpose of the Study:
- To identify and characterize molybdenum-containing proteins in Clostridium pasteurianum during nitrogen fixation.
- To investigate the temporal accumulation of molybdenum and its associated components.
Main Methods:
- Anaerobic multiphasic gel electrophoresis.
- Anion-exchange chromatography.
- 99Mo labeling to trace molybdenum components.
Main Results:
- Molybdenum accumulation precedes nitrogenase activity.
- Six distinct 99Mo-labeled species were identified, including the MoFe protein of nitrogenase, formate dehydrogenase, and a Mo binding-storage protein.
- A soluble molybdenum cofactor was detected, capable of reconstituting nitrate reductase activity.
Conclusions:
- Clostridium pasteurianum actively manages molybdenum uptake and storage.
- Multiple molybdoproteins are involved in nitrogen fixation and related metabolic pathways.
- A free molybdenum cofactor plays a role in enzyme activity and can be transferred between organisms.