Unique features of the nitrogenase VFe protein from Azotobacter vinelandii.
Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N(2)) to ammonia (NH(3)). The vanadium (V)-nitrogenase is very similar to the "conventional" molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of Azotobacter vinelandii V-nitrogenase has been focused on 2 incomplete forms of this protein: alphabeta(2) and alpha(2)beta(2), both of which contain the small delta-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from A. vinelandii. This VFe protein has a previously-unsuspected, alpha(2)beta(2)delta(4)-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein. More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H(2) evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor- and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein.
Lee
Chi Chung
CC
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.
Hu
Yilin
Y
Ribbe
Markus W
MW
D016948:Azotobacter vinelandii / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism; D008983:Molybdoferredoxin / Q000737:chemistry; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014639:Vanadium / Q000378:metabolism
D008983:Molybdoferredoxin; D014639:Vanadium; D006639:Histidine; D009591:Nitrogenase