Two-dimensional hyperfine sublevel correlation spectroscopy: Powder features for S=1/2, I=1

Maryasov, Alexander G.; Bowman, Michael K.

doi:10.1016/j.jmr.2005.11.013

Cited by 11 publications

(6 citation statements)

References 17 publications

(42 reference statements)

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“…This sequence was repeated at a rate of 2 kHz as t 1 and t 2 were independently incremented. 27,28 The signals from 1 H of the axially bound water or from 15 N of PPT were optimized by adjustment of τ . 9 Unless otherwise noted, a value of 240 ns was used for τ .…”

Section: Methodsmentioning

confidence: 99%

“…The echo indicates the measured signal. This sequence was repeated at a rate of 2 kHz as t 1 and t 2 were independently incremented. , The signals from 1 H of the axially bound water or from 15 N of PPT were optimized by adjustment of τ . Unless otherwise noted, a value of 240 ns was used for τ. HYSCORE cross-peaks appear in pairs at coordinates whose frequencies correlate ENDOR transitions of a single nucleus for different values of the heme unpaired electron spin .…”

Section: Methodsmentioning

confidence: 99%

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Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

et al. 2015

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Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP–inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenyl-propyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of “classic” type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A 15N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

et al. 2015

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“…This is a two-dimensional correlation EPR pulsed spectroscopy technique that correlates ENDOR frequencies from the same nucleus. The HYSCORE measurements were performed using a four-pulse sequence, which correlates with the electron nuclear double resonance (ENDOR) frequencies, and disperses the spectrum in two dimensions with increased resolution for small second-order shifts caused by anisotropic interactions − . All HYSCORE spectra were recorded at a magnetic field of 0.2865 T, a nominal microwave frequency of 9.690 GHz, a 90° microwave pulse length of 16 ns, a separation t of 296 ns between the first two microwave pulses, and temperatures between 10 and 11 K. There were 256 points measured in each dimension with an increment of 24 ns between the points.…”

Section: Methodsmentioning

confidence: 99%

“…The anisotropic part of the hyperfine coupling was ascertained indirectly by measuring the second-order shifts in the ENDOR frequencies of protons present in the HYSCORE spectra. These second-order shifts were then separated through a simple linear regression into the principal values of the hyperfine tensor as previously described − .…”

Section: Methodsmentioning

confidence: 99%

Intramolecular Heme Ligation of the Cytochrome P450 2C9 R108H Mutant Demonstrates Pronounced Conformational Flexibility of the B−C Loop Region: Implications for Substrate Binding

Roberts

Cheesman

Primak³

et al. 2010

Biochemistry

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A previous study (i.e. Dickmann, L., et al. (2004) Mol. Pharmacol. 65, 842-850.) revealed some unusual properties of the R108H mutant of cytochrome P450 2C9 (CYP2C9), including elevated thermostability relative to CYP2C9, as well as a UV-visible absorbance spectrum that was indicative of nitrogenous ligation to the heme iron. In the present study, size-exclusion chromatography and UV-visible absorbance spectroscopy of CYP2C9 R108H monomers demonstrated that nitrogen ligation is indeed intramolecular. Pulsed electron paramagnetic resonance of CYP2C9 R108H monomers showed that a histidine is most likely bound to the heme as previously hypothesized. An energy-minimized model of the R108H mutant maintained a CYP fold, despite substantial movement of several loop regions of the mutant and, therefore, represents an extreme example of a closed conformation of the enzyme. Molecular dynamics (MD) simulations of CYP2C9 were performed in order to study the range of energetically accessible CYP2C9 conformations. These in silico studies showed that the B-C loop region of CYP2C9 moves away from the heme to a position resembling the putative open conformation described for rabbit CYP2B4. A model involving the movement of the B-C loop region and R108 between the open and closed conformations of CYP2C9 is presented, which helps to explain the enzyme’s ability to regio- and stereo-specifically metabolize some ligands, while allosterically activating others.

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“…In principle, Eqs. (4)-(6) do not apply for deuterium because this element possesses a nuclear spin I = 1, implying a quadrupolar interaction in addition to hf interaction which strongly influences the shape and intensity of nuclear modulations (Maryasov and Bowman, 2006). However the very small quadrupolar moment of D and the fact that preliminary simulations of HYSCORE spectra show that the quadrupolar interaction is very small (%0.1 MHz) compared to hf interaction (2 MHz) allows us to neglect quadrupolar effects in this case.…”

Section: Figurementioning

confidence: 99%

Extreme deuterium enrichment of organic radicals in the Orgueil meteorite: Revisiting the interstellar interpretation?

Gourier

Robert

Delpoux

et al. 2008

Geochimica et Cosmochimica Acta

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Two-dimensional hyperfine sublevel correlation spectroscopy: Powder features for S=1/2, I=1

Cited by 11 publications

References 17 publications

Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

Intramolecular Heme Ligation of the Cytochrome P450 2C9 R108H Mutant Demonstrates Pronounced Conformational Flexibility of the B−C Loop Region: Implications for Substrate Binding

Extreme deuterium enrichment of organic radicals in the Orgueil meteorite: Revisiting the interstellar interpretation?

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