Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.

We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli. Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His' delta1 protons near 100 ppm, and beta and alpha protons collectively between 20 and 50 ppm, in Fe(III)SOD. In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically. A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the beta protons of Asp 156, superseding earlier assignments (Ming et al. (1994) Inorg. Chem., 33, 83-87). We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of 82 and epsilon1 protons. These are the closest protein protons to Fe(II) and therefore they have the broadest (approximately 4,000 Hz) and most difficult to detect resonances. Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state. Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand of Rhodopseudomonas palustris cytochrome c' (Bertini et al. (1988) Inorg. Chem., 37, 4814-4821 ) suggesting that Fe(II)SOD's equatorial His2Asp- ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+.

Mutagenesis studies of the FeSII protein of Azotobacter vinelandii: roles of histidine and lysine residues in the protection of nitrogenase from oxygen damage.

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.

Mutation of tyrosine 34 to phenylalanine eliminates the active site pK of reduced iron-containing superoxide dismutase.

We have compared the magnetic resonance properties and pH dependence of wild-type and mutant Fe-containing superoxide dismutase (Fe-SOD) in which the conserved active site tyrosine (Tyr 34) is replaced by phenylalanine. The EPR spectrum of the oxidized state and the NMR spectrum of the paramagnetically shifted resonances of the reduced state indicate that in both states the active site is relatively unperturbed by the mutation. Similarly, the mutant Fe-SOD retains approximately 41% of wild-type catalytic activity on a per Fe basis. However, replacement of Tyr 34 by Phe abolishes both NMR spectroscopic signatures of the active site pK of 8.5 of (reduced) Fe2+-SOD. Neither accessibility to base-catalyzed exchange nor the chemical shifts of active site residues are affected by pH in the range of 6.5-10.5 in Y34F Fe2+-SOD. Thus, the active site pK of 8.5 of Fe2+-SOD most likely corresponds to deprotonation of Tyr 34. The widespread chemical shift changes associated with the pK could reflect Tyr 34's participation in the active site hydrogen bonding network and the network's propagation of the effects of deprotonating Tyr 34 to the Fe2+. Deprotonation of Tyr 34 can also explain the dramatic decrease in active site accessibility to base-catalyzed exchange as the result of electrostatic repulsion between the exchange catalyst OH- and the (Tyr 34)- ion formed at high pH. Similar electrostatic repulsion between (Tyr 34)- and the substrate O2.- is also consistent with the observed increase in KM above pH 9.

Spectroscopic measurement of a long-predicted active site pK in iron-superoxide dismutase from Escherichia coli.

The accepted mechanism of Fe-containing superoxide dismutase (Fe-SOD) activity and inhibition by anions implies the existence of a group with a pK of 8.6-9.0 in the active site of reduced Fe-SOD [Bull, C. & Fee, J. A. (1985) J. Am. Chem. Soc. 107, 3295-3304]. We have performed pH titrations of reduced Fe-SOD by NMR spectroscopy and observe a pK of 8.5 at 30 degrees C which is the only pK affecting the active site between pH 5.5 and 10.5. Thus, we present the first spectroscopic evidence of the predicted pK. Although the pK is associated with chemical shift changes for almost all of the resonances of the active site, resonance line widths and the T1 of a ligand proton are not significantly affected by the pK, indicating that there is no significant conformational change and only relatively minor effects on the electronic spin properties of Fe2+. The changes in chemical shift are probably caused by changes in hydrogen bonding to a ligand and attendant subtle perturbation of the Fe2+ paramagnetism upon loss of the proton with the pK of 8.5. The pK is also associated with a dramatic restriction of the exchange of at least one ligand proton. Thus, active site accessibility to solvent and OH- decreases by more than 2 orders of magnitude upon loss of the proton with the pK of 8.5. Since OH- is a competitive inhibitor of Fe-SOD, and thus a substrate analog, this dramatic and unusual decrease in accessibility to OH- is consistent with the increase in the K(M) for O2.- that is associated with a pK near 9.