Construction of a form of the MoFe protein of nitrogenase that accepts electrons from the Fe protein but does not reduce substrate.
The direction of electron flow through nitrogenase is generally believed to be from the Fe protein to the P-clusters to the FeMo cofactor and then to substrate. In order to examine oxidation states of the P-clusters that might be involved in this pathway, we have constructed a form of the MoFe protein that contains a species called the MoFe cluster (Gavini, N., Ma, L., Watt, G., and Burgess, B.K. (1995) Biochemistry 33, 11842-11849) in place of FeMo cofactor. This MoFe cluster-containing protein was purified, and the presence of the cluster was confirmed by reisolation of the MoFe cluster followed by EPR spectroscopy. The protein does not reduce protons or acetylene, however, upon the addition of the Fe protein and MgATP, MgATP hydrolysis occurs at a rate 28% of the wild-type protein. As isolated in the presence of excess dithionite the MoFe cluster-containing protein is EPR silent. Upon addition of the Fe protein and MgATP a g = 1.94 EPR signal develops that integrates to about 1 spin per P-cluster. This signal only develops when both the Fe protein and MgATP are added and it arises from the P-clusters.
Ma
L
L
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717-3900, USA.
Brosius
M A
MA
Burgess
B K
BK
D016948:Azotobacter vinelandii / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D008983:Molybdoferredoxin; D009591:Nitrogenase