Purification and characterization of a FeMo cofactor-deficient MoFe protein.
Previous studies have shown that the nifH gene product is required for FeMo cofactor biosynthesis and insertion and that a delta nifH strain of Azotobacter vinelandii designated DJ54 accumulates a FeMo cofactor-deficient MoFe protein that is distinct from the FeMo cofactor-deficient protein synthesis by Nif B-, N-, or E- strains [Tal, S., Chun, T., Gavini, N., & Burgess, B. K. (1991) J. Biol. Chem. 266, 10654-10657]. Here we report the purification and activation of the MoFe protein from DJ54. The purified protein is an alpha 2 beta 2 tetramer that is indistinguishable from the wild-type MoFe protein by the criteria of SDS-polyacrylamide gel electrophoresis, native gel electrophoresis, and two-dimensional gel electrophoresis. It binds normally to its redox partner, the Fe protein, by the criterion of chemical cross-linking. It does not contain FeMo cofactor and does not catalyze significant C2H2 reduction or reduction-independent MgATP hydrolysis. It can, however, be activated with FeMo cofactor following the addition of the Fe protein and MgATP when an additional required component(s) is supplied by cell-free extracts from a delta nifD strain of A. vinelandii. The purified DJ54 MoFe protein does contain P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the POX state. In the presence of dithionite it exhibits an axial S = 1/2 EPR signal that integrates to 0.1-0.3 spin per alpha 2 beta 2 tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)
Gavini
N
N
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Ma
L
L
Watt
G
G
Burgess
B K
BK
D000255:Adenosine Triphosphate / Q000378:metabolism; D001395:Azotobacter; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005030:Ethylenes / Q000378:metabolism; D017353:Gene Deletion; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008982:Molybdenum / Q000032:analysis; D008983:Molybdoferredoxin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009591:Nitrogenase / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D013053:Spectrophotometry
D001426:Bacterial Proteins; D005030:Ethylenes; D007506:Iron-Sulfur Proteins; D008983:Molybdoferredoxin; D008982:Molybdenum; D000255:Adenosine Triphosphate; C036216:ethylene; D007501:Iron; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase