期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 126, 期 39, 页码 12477-12491出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja0482583
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资金
- NIGMS NIH HHS [GM64631] Funding Source: Medline
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 (N-3-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 (MnSOD)-S-3divided by (possessing a five-coordinate Mn3+ center) and Y34F N-3-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 N-3-(MnSOD)-S-3divided by at room temperature (RT). Comparison of excited-state spectroscopic data reveals that Mn3+SOD and RT N-3-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 N-3-Mn3+SOD and Y34F N-3-Mn3+SOD at 0 degreesC 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 N-3-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-Mn3+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 N-3-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.
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