Mutagenesis Studies of the FeSII Protein of Azotobacter vinelandii: Roles of Histidine and Lysine Residues in the Protection of Nitrogenase from Oxygen Damage

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• 作者：Jianrong Lou ; Farhad Moshiri ; Michael K. Johnson ; Meghan E. Lafferty ; David L. Sorkin ; A.-F. Miller ; and Robert J. Maier
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：April 27, 1999
• 年：1999
• 卷：38
• 期：17
• 页码：5563 - 5571
• 全文大小：103K
• 年卷期：v.38,no.17(April 27, 1999)
• ISSN：1520-4995

文摘

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complexwith nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for itsaction is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSIIprotein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein wereport the construction and characterization of five site-directed mutants of the FeSII protein (H12Q,H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after beingindividually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assemblyand orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization andtheir redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities ofthe individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenaseactivities after adding each pure version back to extracts from an FeSII deletion strain, and then exposingthe mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein.However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much lessefficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states,which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsiveto oxidation state, and the pK increase of 0.16 pH unit upon 2Fe-2S cluster oxidation is indicative ofionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSIIprotein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSIIproteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all itsprotective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysinemutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild typestrain under carbon substrate-limited conditions. These results indicate that the two lysines may have animportant role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase componentproteins.