Abstract
| - A variety of spectroscopic and computational techniques have been used to examine thethermochromic transition previously reported for the oxidized state of Mn-dependent superoxide dismutasefrom E. coli in the presence of substrate analog azide (N3−Mn3+SOD).[Whittaker, M. M.; Whittaker, J. W.Biochemistry1996, 35, 6762−6770.] Although previous spectroscopic studies had shown that thisthermochromic event corresponds to a change in coordination number of the active-site Mn3+ ion from 6to 5 as temperature is increased, the ligand that dissociates in this conversion had yet to be identified.Through the use of electronic absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies,both d→d and ligand-to-metal charge-transfer (LMCT) transition energies have been determined for nativeMn3+SOD (possessing a five-coordinate Mn3+ center) and Y34F N3−Mn3+SOD (forming a six-coordinateN3−Mn3+ adduct at all temperatures). These two systems provide well-defined reference points from whichto analyze the absorption and CD data obtained for N3−Mn3+SOD at room temperature (RT). Comparisonof excited-state spectroscopic data reveals that Mn3+SOD and RT N3−Mn3+SOD exhibit virtually identicald→d transition energies, suggesting that these two species possess similar geometric and electronicstructures and, thus, that azide does not actually coordinate to the active-site Mn3+ ion at RT. However,resonance Raman spectra of both N3−Mn3+SOD and Y34F N3−Mn3+SOD at 0 °C exhibit azide-relatedvibrations, indicating that azide does interact with the active site of the native enzyme at this temperature.To gain further insight into the nature of the azide/Mn3+ interaction in RT N3−Mn3+SOD, several viableactive-site models designed to promote either dissociation of coordinated solvent, Asp167, or azide weregenerated using DFT computations. By utilizing the time-dependent DFT method to predict absorptionspectra for these models of RT N3−Mn3+SOD, we demonstrate that only azide dissociation is consistentwith experimental data. Collectively, our spectroscopic and computational data provide evidence that theactive site of N3−Mn3+SOD at RT exists in a dynamic equilibrium, with the azide molecule either hydrogen-bonded to the second-sphere Tyr34 residue or coordinated to the Mn3+ ion. These results further highlightthe role that second-sphere residues, especially Tyr34, play in tuning substrate (analog)/metal ioninteractions.
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