Isolation of two forms of the nitrogenase VFe protein from Azotobacter vinelandii.
When Q-Sepharose was used in the purification of the V nitrogenase proteins from Azotobacter vinelandii, an increase in resolution was observed that resulted in a separation of the nitrogenase component 1 protein (Av1') into two forms, labeled Av1'A and Av1'B. Even though both forms possessed the same enzymatic behavior, Av1'A exhibited a lower specific activity and migrated during gel filtration with an apparent lower molecular weight than Av1'B. Furthermore, SDS-polyacrylamide gel electrophoresis showed different relative compositions of the two major subunits of both forms, with Av1'A possessing a trimer (alpha beta 2) pattern compared to the more typical tetramer (alpha 2 beta 2) pattern found for Av1'B. Metal analysis indicated a V-to-Fe ratio of 1:19 for Av1'A and 1:15 (or 2:30) for Av1'B, while acid-labile sulfide analysis showed that Av1'A possessed about half as much sulfide as Av1'B. EPR spectroscopy revealed that both proteins retained the S = 3/2 and S = 1/2 signals observed in earlier isolations, with an additional S = 1/2 signal present in the spectrum of protein A. These results suggest that Av1'A is an incomplete form of the VFe protein, containing only one cofactor and one P cluster with an additional [Fe4-S4]-like cluster. The presence of a V storage protein in A. vinelandii was also investigated. Although no V storage protein was found, two V-binding proteins were observed.
Blanchard
C Z
CZ
Louisiana State University, Baton Rouge 70803-1804, USA.
Hales
B J
BJ
D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification; D002352:Carrier Proteins / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000032:analysis; D015394:Molecular Structure; D008970:Molecular Weight; D009591:Nitrogenase / Q000737:chemistry / Q000302:isolation & purification*; D011487:Protein Conformation; D014639:Vanadium / Q000032:analysis
D001426:Bacterial Proteins; D002352:Carrier Proteins; D014639:Vanadium; D007501:Iron; D009591:Nitrogenase