A combined spectroscopic/computational approach has been utilized to explore the chemical origins of the active-site pKs of the structurally homologous Fe- and Mn-dependent superoxide dismutases (SODs). Absorption, circular dichroism, magnetic circular dichroism, and variable-temperature, variable-field magnetic circular dichroism spectroscopic experiments have permitted us to determine electronic transition energies and polarizations, as well as ground-state spin Hamiltonian parameters. These experimental data have been used in conjunction with semiempirical intermediate neglect of differential overlap/spectroscopic parametrization configuration interaction (INDO/S-CI) computations for evaluating hypothetical active-site models for the high-pH species generated by density functional theory (DFT) geometry optimizations. Our experimental and computational data indicate that both reduced FeSOD and oxidized MnSOD do not bind hydroxide at high pH; rather, the active-site pK for these two species is attributed to deprotonation of a second-sphere tyrosine. Conversely, our data obtained on oxidized FeSOD indicate that hydroxide binding is responsible for the observed active-site pK for this species. Intriguingly, in the Fe-substituted form of MnSOD this identical chemical event occurs at a significantly lower pH. Overall, our results suggest an important role for second-sphere amino acids in tuning the active sites' interaction with small anions and bring into question the assumption that these homologous enzymes operate by the same molecular mechanism.

The highly homologous proteins of Fe-containing superoxide dismutase (FeSOD) and MnSOD from Escherichia coli nonetheless exert very different redox tuning on the active site metal ion [Vance; Miller J. Am. Chem. Soc. 1998, 120, 461-467; Biochemistry 2001, 40, 13079-13087]. This was proposed to stem from different hydrogen bonding between the protein and the metal ion's coordinated solvent molecule, and the tight coupling between the protonation state of coordinated solvent and the oxidation state of the metal ion. We now present density functional theory (DFT) calculations on Fe2+ and Fe3+ bound to models of both FeSOD and MnSOD. The calculations support a very important role for the conserved second sphere Gln in MnSOD in specifically destabilizing coordinated H2O relative to coordinated OH-, and thus disfavouring the oxidized state of the metal ion. To test these results we have mutated this Gln to Glu, which is isosteric and isoelectronic to Gln but functions as an H-bond acceptor instead of an H-bond donor and thus should increase the stability of Fe2+-bound H2O. In accordance with the calculations, Q69E-FeSOD displays a significantly higher reduction potential than wild-type FeSOD. Thus we have demonstrated that hydrogen bonds to coordinated solvent can exert strong redox tuning on a metal ion.

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A combination of spectroscopic and computational methods has been employed to explore the nature of the yellow and pink low-temperature azide adducts of iron(III) superoxide dismutase (N(3)-FeSOD), which have been known for more than two decades. Variable-temperature variable-field magnetic circular dichroism (MCD) data suggest that both species possess similar ferric centers with a single azide ligand bound, contradicting previous proposals invoking two azide ligands in the pink form. Complementary data obtained on the azide complex of the Q69E FeSOD mutant reveal that relatively minor perturbations in the metal-center environment are sufficient to produce significant spectral changes; the Q69E N(3)-FeSOD species is red in color at all temperatures. Resonance Raman (RR) spectra of the wild-type and Q69E mutant N(3)-FeSOD complexes are consistent with similar Fe-N(3) units in all three species; however, variations in energies and relative intensities of the RR features associated with this unit reveal subtle differences in (N(3)(-))-Fe(3+) bonding. To understand these differences on a quantitative level, density functional theory and semiempirical INDO/S-CI calculations have been performed on N(3)-FeSOD models. These computations support our model that a single azide ligand is present in all three N(3)-FeSOD adducts and suggest that their different appearances reflect differences in the Fe-N-N bond angle. A 10 degrees increase in the Fe-N-N bond angle is sufficient to account for the spectral differences between the yellow and pink wild-type N(3)-FeSOD species. We show that this bond angle is strongly affected by the second coordination sphere, which therefore might also play an important role in orienting incoming substrate for reaction with the FeSOD active site.

The shape of the spectral features in arrival time distributions (ATDs) recorded by ion mobility spectrometry (IMS) can often be interpreted in terms of the coexistence of different isomeric species. Interconversion between such species is also acknowledged to influence the shape of the ATD, even if no general quantitative description of this effect is available. We present an analytical model that allows simulating ATDs resulting from interconverting species. This model is used to reproduce experimental data obtained on a bistable system and to interpret discrepancies between measurements on different types of instruments. We show that the proposed model can be further exploited to extract kinetic and thermodynamic data from tandem-IMS measurements.

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Two N-pyrenylacetamide-substituted sugar-aza-crown ethers have been synthesized as new fluorescent chemosensors. The designed ligands 1 and 2 exhibit fluorescence characteristics of a pyrene monomer and a dynamic excimer emission when compared to N-pyrenylacetamide as a model compound. Both ligands displayed a Cu2+-sensitive fluorescence quenching with a 1:1 stoichiometry and high stability constants (log K = 6.7 for 1 and 7.8 for 2). The quenching effect was rationalized on the basis of photoinduced electron transfer from the excited pyrene to the complexed Cu2+ cation, while the changes in excimer-to-monomer ratio were explained by a conformational analysis through DFT calculations. The predicted structure suggests that the Cu2+ cation is coordinated with the two carbonyl groups and the sugar-aza-crown ethers which rigidified the complex structure and placed the two pyrene moieties far apart.