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

Abstract

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report 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 being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the 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 less efficient 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 responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.

Evidence for Conformational Protection of Nitrogenase against Oxygen in Gluconacetobacter diazotrophicus by a Putative FeSII Protein

“…Conformational protection is due to the formation of a complex between the FeSII (Shethna) protein and nitrogenase under high intracellular oxygen concentrations. In this complex, nitrogenase is inactive but transiently protected from damage by oxygen (10,13,21). Additional studies have shown that the function of the FeSII protein is of major importance for cell viability under carbon and/or energy limitation but only when cells are grown diazotrophically (12,14).…”

Protection from superoxide damage associated with an increased level of the YggX protein in Salmonella enterica

“…A distinct way of protecting [Fe-S] clusters is shown by the FeSII protein of Azotobacter vinelandii. The FeSII, or Shetna protein, forms a complex with nitrogenase under periods of high oxygen exposure, thus protecting the essential [Fe-S] cluster from oxidation (16,17).…”

Expression of a Heterologous Manganese Superoxide Dismutase Gene in Intestinal Lactobacilli Provides Protection against Hydrogen Peroxide Toxicity

“…Figure 6 shows that the cells have redundant mechanisms for protection against the toxicity of H 2 O 2 . Thus, the protection against H 2 O 2 and against the generation of HO˙can be accomplished by (i) elimination of O 2 ·Ϫ by SODs to inhibit reactions 1 and 3; (ii) elimination of H 2 O 2 by hydroperoxidases to inhibit reaction 2; (iii) chelation of free iron by iron-chelator or by specific DNA-binding proteins (e.g., Dpr and Dps in gram-positive and gram-negative organisms, respectively) (27,59) to inhibit reaction 2; (iv) use of specific [Fe-S]cluster protectors-e.g., (20) and the Shetna protein (FeSII) in Azotobacter vinelandii (35) to inhibit reaction 1; or (v) the repair of damaged DNA and other macromolecules (8). Data in Fig.…”