The FeMoco-deficient MoFe protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster features.
The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (Delta(nifH) MoFe protein) was purified in large quantity. The alpha(2)beta(2) tetrameric Delta(nifH) MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The Delta(nifH) MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g approximately 2 region. The indigo disulfonate-oxidized Delta(nifH) MoFe protein does not show features of the P(2+) state of the P-cluster of the Delta(nifB) MoFe protein. The oxidized Delta(nifH) MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S](1+) cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S](1+) cluster of the Fe protein. Furthermore, the dithionite-reduced Delta(nifH) MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.
Ribbe
Markus W
MW
Department of Molecular Biology and Biochemistry and the Program in Macromolecular Structure, University of California, Irvine, California 92697-3900, USA. markus@nitrolab.bio.uci.edu
Hu
Yilin
Y
Guo
Maolin
M
Schmid
Benedikt
B
Burgess
Barbara K
BK
D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry; D019343:Citric Acid / Q000494:pharmacology; D008983:Molybdoferredoxin / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*
D001426:Bacterial Proteins; D008983:Molybdoferredoxin; C092498:NifB protein, Bacteria; C419387:titanium citrate; D019343:Citric Acid; D010088:Oxidoreductases; C029130:nitrogenase reductase