Toward modeling the high chloride, low pH form of sulfite oxidase: Ka-band ESEEM of equatorial chloro ligands in oxomolybdenum(V) complexes

Astashkin, Andrei V.; Klein, Eric L.; Enemark, John H.

doi:10.1016/j.jinorgbio.2007.05.015

Cited by 19 publications

(24 citation statements)

References 37 publications

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“…DFT calculations for Cl clearly demonstrated the unfeasibility of Cl − coordination in the otherwise vacant axial position, contrary to the assumptions made by George and coworkers10 and also by us initially 46. The calculations showed that Cl − is most likely located in the second coordination sphere (as in PDB 2A99)4 and that this structure gives the best agreement of the derived and experimental spectroscopic parameters 11.…”

Section: Discussioncontrasting

confidence: 57%

Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for “difficult” nuclei

et al. 2011

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The catalytic mechanisms of sulfite oxidizing enzymes (SOEs) have been investigated by multi-frequency pulsed EPR measurements of “difficult” magnetic nuclei (35,37Cl, 33S, 17O) associated with the Mo(V) center. Extensive DFT calculations have been used to relate the experimental magnetic resonance parameters of these nuclei to specific active site structures. This combined spectroscopic and computational approach has provided new insights concerning the structure/function relationships of the active sites of SOEs, including: (i) the exchange of oxo ligands; (ii) the nature of the blocked forms; and (iii) the role of Cl− in low pH forms.

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Section: Discussioncontrasting

confidence: 57%

Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for “difficult” nuclei

et al. 2011

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“…Also, the nqi estimated in this work (~ 3 MHz) is about one order of magnitude smaller than the nqi constants previously obtained for covalently bound Cl − . In model complexes containing the cis -oxo-Mo(V)-Cl fragment, Cl − has nqi constants of tens of MHz 18, 46. This comparison eliminates any possibility of inner-sphere equatorial coordination of Cl − to Mo(V) in lpH SO, in agreement with the available structural knowledge for the molybdenum center of SO discussed in the Introduction.…”

Section: Resultsmentioning

confidence: 99%

“…These results unequivocally and directly confirm the presence of Cl − in close proximity to the Mo(V) center of the lpH form of SO and provide the first reliable hyperfine ( hfi ) and nuclear quadrupole ( nqi ) interaction parameters for the chlorine nucleus. The structural implications of the parameters obtained are discussed using density functional theoretical (DFT) calculations, spectroscopic data from model oxomolybdenum compounds,18 and the available general structural information related to the molybdenum center and its protein environment.…”

Section: Introductionmentioning

confidence: 99%

Direct Detection and Characterization of Chloride in the Active Site of the Low-pH Form of Sulfite Oxidase Using Electron Spin Echo Envelope Modulation Spectroscopy, Isotopic Labeling, and Density Functional Theory Calculations

et al. 2009

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Electron spin echo envelope modulation (ESEEM) investigations were carried out on samples of the low-pH (lpH) form of vertebrate sulfite oxidase (SO) prepared with 35 Cl-and 37 Cl-enriched buffers as well as with buffer containing the natural abundance of Cl isotopes. The isotope-related changes observed in the ESEEM spectra provide direct and unequivocal evidence that Cl − is located in close proximity to the Mo(V) center of lpH SO. The measured isotropic hyperfine interaction constant of about 4 MHz ( 35 Cl) suggests that the Cl − ion is either weakly coordinated to Mo(V) at its otherwise vacant axial position, trans to the oxo ligand, or is hydrogen-bonded to the equatorial exchangeable OH ligand. Scalar relativistic all-electron density functional theory (DFT) calculations of the hyperfine and nuclear quadrupole interaction parameters, along with steric and energetic arguments, strongly support the possibility that Cl − is hydrogen-bonded to the equatorial OH ligand rather than being directly coordinated to the Mo(V).

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“…a SAD, b NITRATE, c PERCHLORATE, and d CYANIDE, The2mFo-DFc electron density maps contoured at 1r (blue) and the mFo-DFc electron density maps contoured at 3r (red) calculated in the absence of a sixth ligand (left) and calculated in the presence of the sulfur atom in the final refinement (right) g n * 0.5 for 35,37 Cl and I = 1/2, g n = 2.26 for 31 P) and hence superhyperfine structure from these nuclei might be observed in the EPR spectra associated with Mo(V) species. Hyperfine couplings associated with chloride nuclei in equatorial positions have been reported in model complexes of Mo(V) ions in nearly octahedral or squarepyramidal coordination [24] and in the molybdenum enzyme sulfite oxidase [25]. Larger couplings should be expected for the phosphorous atom because of the higher nuclear g factor (g n = 2.26).…”

Section: Analysis Of the Structures Around The Molybdenum Active Sitementioning

confidence: 96%

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

et al. 2008

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Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 Å . The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH 2 ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (k = 1.77 Å ) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sc atom of the molybdenum ligand Cys A140 was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 Å , indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with 15 N-labeled nitrate and in D 2 O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligandbased redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

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Toward modeling the high chloride, low pH form of sulfite oxidase: Ka-band ESEEM of equatorial chloro ligands in oxomolybdenum(V) complexes

Cited by 19 publications

References 37 publications

Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for “difficult” nuclei

Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for “difficult” nuclei

Direct Detection and Characterization of Chloride in the Active Site of the Low-pH Form of Sulfite Oxidase Using Electron Spin Echo Envelope Modulation Spectroscopy, Isotopic Labeling, and Density Functional Theory Calculations

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum

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