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PH-dependent inhibition by azide and fluoride of the iron superoxide dismutase from Propionibacterium shermanii.

ABSTRACT: The iron-containing superoxide dismutase from Propionibacterium shermanii shows, in contrast with other iron superoxide dismutases, only a minor inhibition by azide or fluoride (10-100 mM) of up to 23% at pH 7.8. The activity of the protein with Mn bound to the active site was not diminished under the same conditions. The binding constant between azide and the Fe3+ ion was determined as approx. 2 mM and for fluoride approx. 2.3 mM; they are so far comparable to those known for other iron superoxide dismutases. This seems to be a discrepancy because all other iron superoxide dismutases so far known are described as being inhibited by 50-70% by 10 mM azide. However, towards lower pH there was a drastically increased inhibition by both anions. At pH 6.8 about 80% inhibition was exhibited by azide or fluoride at a concentration of 10 mM or higher. In contrast, on increasing the pH, azide or fluoride still bound to the Fe3+ at the active site but their inhibition capacity decreased. This observation implies that both anions bind to the metal at a position that is empty at low pH, whereas at higher pH water or a negatively charged hydroxyl anion is bound. It is likely that the superoxide anion binds to the same position and has to replace the sixth ligand, leading to a diminished catalytic activity of the superoxide dismutase owing to steric and/or electrostatic inhibition of the ligand.

SUBMITTER: Meier B

PROVIDER: S-EPMC1219368 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

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Project description:We have synthesized and characterized, using X-ray crystallographic, spectroscopic, and computational techniques, a six-coordinate diazide Fe (3+) complex, LFe(N 3) 2 (where L is the tetradentate ligand 7-diisopropyl-1,4,7-triazacyclononane-1-acetic acid), that serves as a model of the azide adducts of Fe (3+) superoxide dismutase (Fe (3+)SOD). While previous spectroscopic studies revealed that two distinct azide-bound Fe (3+)SOD species can be obtained at cryogenic temperatures depending on protein and azide concentrations, the number of azide ligands coordinated to the Fe (3+) ion in each species has been the subject of some controversy. In the case of LFe(N 3) 2, the electronic absorption and magnetic circular dichroism spectra are dominated by two broad features centered at 21 500 cm (-1) (approximately 4000 M (-1) cm (-1)) and approximately 30 300 cm (-1) (approximately 7400 M (-1) cm (-1)) attributed to N3 (-) --> Fe (3+) charge transfer (CT) transitions. A normal coordinate analysis of resonance Raman (RR) data obtained for LFe(N 3) 2 indicates that the vibrational features at 363 and 403 cm (-1) correspond to the Fe-N 3 stretching modes (nu Fe-N3) associated with the two different azide ligands and yields Fe-N 3 force constants of 1.170 and 1.275 mdyne/A, respectively. RR excitation profile data obtained with laser excitation between 16,000 and 22,000 cm (-1) reveal that the nu Fe-N3 modes at 363 and 403 cm (-1) are preferentially enhanced upon excitation in resonance with the N 3 (-) --> Fe (3+) CT transitions at lower and higher energies, respectively. Consistent with this result, density functional theory electronic structure calculations predict a larger stabilization of the molecular orbitals of the more strongly bound azide due to increased sigma-symmetry orbital overlap with the Fe 3d orbitals, thus yielding higher N 3 (-) --> Fe (3+) CT transition energies. Comparison of our data obtained for LFe(N 3) 2 with those reported previously for the two azide adducts of Fe (3+)SOD provides compelling evidence that a single azide is coordinated to the Fe (3+) center in each protein species.

Project description:We have prepared and thoroughly characterized, using X-ray crystallographic, spectroscopic, and computational methods, the diazide adduct of [Fe(III)(dapsox)(H2O)2](+) [dapsox = 2,6-diacetylpyridinebis(semioxamazide)], (1), a low-molecular weight, functional analogue of iron superoxide dismutase (FeSOD). The X-ray crystal structure of the dimeric form of 1, (Na[Fe(III)(dapsox)(N3)2]·DMF)2 (2) shows two axially coordinated, symmetry inequivalent azides with differing Fe-N3 bond lengths and Fe-N-N2 bond angles. This inequivalence of the azide ligands likely reflects the presence of an interdimer hydrogen bonding interaction between a dapsox NH group and the coordinated nitrogen of one of the two azide ligands. Resonance Raman (rR) data obtained for frozen aqueous solution and solid-state samples of 2 indicate that the azides remain inequivalent in solution, suggesting that one of the azide ligands of 1 engages in an intermolecular hydrogen bonding interaction with a water molecule. Density functional theory (DFT) and time-dependent DFT calculations have been used to study two different computational models of 1, one using coordinates taken from the X-ray crystal structure of 2, and the other generated via DFT geometry optimization. An evaluation of these models on the basis of electronic absorption, magnetic circular dichroism, and rR data indicates that the crystal structure based model yields a more accurate electronic structure description of 1, providing further support for the proposed intermolecular hydrogen bonding of 1 in the solid state and in solution. An analysis of the experimentally validated DFT results for this model reveals that the azides have both ?- and ?-bonding interactions with the Fe(III) center and that more negative charge is located on the Fe-bound, rather than on the terminal, nitrogen atom of each azide. These observations are reminiscent of the results previously reported for the azide adduct of FeSOD and provide clues regarding the origin of the high catalytic activity of Fe-dapsox for superoxide disproportionation.

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PH-dependent inhibition by azide and fluoride of the iron superoxide dismutase from Propionibacterium shermanii.

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