Nickel Superoxide Dismutase: Structural and Functional Roles of His1 and Its H-Bonding Network

Abstract

Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the N-H of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intra-subunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network, and compare the results with prior predictions from DFT calculations. H1A-NiSOD, which lacks the apical ligand entirely, was crystallographically characterized and reveals that in the absence of the Glu17-His1 H-bond, the active site is disordered. Subsequent characterization using X-ray absorption spectroscopy (XAS) shows that Ni(II) is bound in the expected N2S2 planar coordination site. Despite these structural perturbations, the H1A-NiSOD variant is an active catalyst with 4% of WT-NiSOD activity. Three other mutations were designed to preserve the apical imidazole ligand, but perturb the H-bonding network: R47A-NiSOD, lacks the intra-molecular H-bonding interaction, E17R/R47A-NiSOD, which retains the intra-molecular H-bond, but lacks the inter-molecular Glu17-His1 H-bond, and E17A/R47A-NiSOD, which lacks both H-bonding interactions. These variants were characterized by a combination of techniques including XAS characterization of the nickel site structure, kinetic studies employing pulse-radiolytic production of superoxide, and EPR and chemical probes of the redox activity. The results indicate that in addition to the roles in redox tuning suggested by the computational models, the Glu17-His1 H-bond plays an important structural role in the formation of the Ni-hook motif that is a critical feature of the active site.

Spectroscopically Characterized Synthetic Mononuclear Nickel–Oxygen Species

“…Superoxonickel species are proposed to be key reactioni ntermediates in the catalytic cycle of nickel superoxide dismutase. [27,28] This is also ap lausible formulation for the nickel-dioxygen speciesw hich was recently crystallographically characterizedi nn ickel-dependent quercetin 2,4-dioxygenase ( Figure 1). [7] In 2004 Riordana nd co-workersr eported the formation of as ide-on superoxonickel(II) species by reactiono ft he nickel(I) complex [Ni I (PhTt Ad )(CO)] with O 2 .…”

A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site

“…The role of this hydrogen-bonding network was studied by altering this network using H1A, R47A, E17A/R47A, E17R/R47E mutants. 72 Metal analysis using ICP-OES showed that these mutants are still capable of binding nickel; and EXAFS analysis showed that the Ni binds in the same site in these mutants as in WT-NiSOD. The mutants displayed decreased thermal stability, which is more pronounced for the ones with altered His1/Glu17 hydrogen bonding.…”

“…The mutants displayed decreased thermal stability, which is more pronounced for the ones with altered His1/Glu17 hydrogen bonding. 72 The mutants that are EPR active (R47A and E17R/R47E) displayed similar hyperfine splitting at the g z peak indicating that the mutants retain the His-on structure. However, the EPR intensities in these two mutants are significantly decreased compared to WT-NiSOD with the intensities being ~15% for R47A and ~8% for E17R/R47E.…”

“…In a recently published result, H1A-NiSOD showed a similar decrease in catalytic activity and displayed a unique spectral behavior (Figure 1.10), suggesting a transient species during catalysis that is not observed in WT-NiSOD. 72 Kinetics employing pulse radiolytic generation of O 2 and reduced WT-NiSOD showed the rapid appearance of the absorption maximum at ~380 nm that has been assigned to S Cys →Ni(III) charge transfer and its persistence during subsequent turnovers. In the H1A- NiSOD mutant, however, the spectral behavior shows a rapid increase in absorbance at ~390 nm followed by a decay that is much slower than catalysis.…”

“…•-] and occurs in two steps, one with a rate constant k = 40 s -1 and the other at k = 3 s -1 . 72 (1)A)] showed an increased peak to peak separation as the scan rate is increased from 5 mV/s to 50 V/s. The voltammogram displayed quasireversible behavior and the peak separation, as a function of scan rate, has an exponential behavior.…”