Ribbe, Markus W. and Hu, Yilin and Guo, Maolin and Schmidt, Benedikt and Burgess, Barbara K. (2002) The FeMoco-deficient MoFe Protein Produced by a nifH Deletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features. Journal of Biological Chemistry, 277 (26). pp. 23469-23476. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:RIBjbc02
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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 2beta 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 approx 2 region. The indigo disulfonate-oxidized Delta nifH MoFe protein does not show features of the P2+ 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.
|Additional Information:||Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, March 1, 2002, and in revised form, April 23, 2002. Originally published In Press as doi:10.1074/jbc.M202061200 on April 26, 2002. We acknowledge Professor Dennis Dean of Virginia Polytechnic Institute and State University for kindly providing the Delta nifH A. vinelandii strain DJ1165, Professor Douglas Rees of the California Institute of Technology (Pasadena) for generous support of EPR analysis, Farzad Naderi (University of California, Irvine, program of biotechnology) for technical assistance, and Professor Tom Poulos and Professor Jerry Manning of the University of California, Irvine, for enormous support through the writing of this paper. This work was supported by National Institutes of Health Grant GM-43144 (to B. K. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We dedicate this paper to Professor Barbara K. Burgess for her ingenious contribution to this work. [B.K.B.] Deceased December 29, 2001.|
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|Deposited On:||12 Jun 2008|
|Last Modified:||26 Dec 2012 10:05|
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