Spectroscopic and computational studies of the azide-adduct of manganese superoxide dismutase: Definitive assignment of the ligand responsible for the low-temperature thermochromism

Timothy A. Jackson, Anush Karapetian, Anne Frances Miller, Thomas C. Brunold

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51 Scopus citations


A variety of spectroscopic and computational techniques have been used to examine the thermochromic transition previously reported for the oxidized state of Mn-dependent superoxide dismutase from E. coli in the presence of substrate analog azide (N3-Mn3+SOD).[Whittaker, M. M.; Whittaker, J. W. Biochemistry 1996, 35, 6762-6770.] Although previous spectroscopic studies had shown that this thermochromic event corresponds to a change in coordination number of the active-site Mn3+ ion from 6 to 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 native Mn3+SOD (possessing a five-coordinate Mn3+ center) and Y34F N3-Mn3+SOD (forming a six-coordinate N 3-Mn3+ adduct at all temperatures). These two systems provide well-defined reference points from which to analyze the absorption and CD data obtained for N3-Mn3+SOD at room temperature (RT). Comparison of excited-state spectroscopic data reveals that Mn3+SOD and RT N3-Mn3+SOD exhibit virtually identical d→d transition energies, suggesting that these two species possess similar geometric and electronic structures 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-related vibrations, 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 viable active-site models designed to promote either dissociation of coordinated solvent, Asp167, or azide were generated using DFT computations. By utilizing the time-dependent DFT method to predict absorption spectra for these models of RT N3-Mn 3+SOD, we demonstrate that only azide dissociation is consistent with experimental data. Collectively, our spectroscopic and computational data provide evidence that the active 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 highlight the role that second-sphere residues, especially Tyr34, play in tuning substrate (analog)/metal ion interactions.

Original languageEnglish
Pages (from-to)12477-12491
Number of pages15
JournalJournal of the American Chemical Society
Issue number39
StatePublished - Oct 6 2004

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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