Isolation of an iron-molybdenum cofactor from nitrogenase.
A method for the isolation of an iron-molybdenum cofactor (FeMoCo) from component I of nitrogenase is described. This method is used to isolate FeMoCo from aerobic, anaerobic, facultative, and photosynthetic nitrogen-fixing organisms. The Fe/Mo ratio in the FeMoCo from Azotobacter vinelandii and Clostridium pasteurianum is 8:1. The FeMoCo contains six atoms of acid-labile sulfide per eight Fe atoms. Crystalline component I from A. vinelandii contains 2 Mo, 33 Fe, and 27 acid-labile sulfide atoms per molecular weight of 250,000. The specific activity of FeMoCo is 425 nmol of C(2)H(4) formed/min per nmol of Mo. There is better than 98% reconstitution between FeMoCo and inactive component I in A. vinelandii mutant strain UW45. The FeMoCo yield from component I is about 90%. FeMoCo from nitrogenase component I of C. pasteurianum, Klebsiella pneumoniae, Bacillus polymyxa, and Rhodospirillum rubrum activates inactive component I in an extract from mutant strain UW45 and follows saturation kinetics. The FeMoCo in various nitrogen-fixing organisms seems to be very similar. Wild-type A. vinelandii derepressed for nitrogenase synthesis in tungsten-containing medium and K. pneumoniae mutant strain UN109 are also activated in vitro by FeMoCo. The FeMoCo is very sensitive to oxygen, but is stable even at room temperature as long as it is kept anaerobic and in N-methylformamide, the solvent used for its isolation. FeMoCo is unstable in an aqueous environment, even though it is kept strictly anaerobic. Knowledge of the structure of this cofactor should be useful for understanding the role of molybdenum at the active site of nitrogenase, role of ligands close to molybdenum in electron and proton transfer, and the catalytic mechanism of nitrogen fixation. The FeMoCo might be used as a model for synthesizing catalysts for chemical nitrogen fixation.
D000332:Aerobiosis; D000693:Anaerobiosis; D001395:Azotobacter / Q000201:enzymology; D001407:Bacillus / Q000201:enzymology; D003013:Clostridium / Q000201:enzymology; D003067:Coenzymes / Q000302:isolation & purification* / Q000494:pharmacology; D004789:Enzyme Activation; D007501:Iron / Q000032:analysis*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008982:Molybdenum / Q000032:analysis*; D009154:Mutation; D009591:Nitrogenase / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology; D013053:Spectrophotometry
D003067:Coenzymes; D008982:Molybdenum; D007501:Iron; D009591:Nitrogenase