UA cloudflare authentication

Browsing by Author "Vennam, Preethi"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    1,2,3-Triazole-Heme Interactions in Cytochrome P450: Functionally Competent Triazole-Water-Heme Complexes
    (American Chemical Society, 2012) Conner, Kip P.; Vennam, Preethi; Woods, Caleb M.; Krzyaniak, Matthew D.; Bowman, Michael K.; Atkins, William M.; University of Washington; University of Washington Seattle; University of Alabama Tuscaloosa
    In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ), the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among cytochrome P450 (CYP) inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via "click" chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and density functional theory computational studies were performed with unsubstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent-1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme-ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism-based inactivator, 17-alpha-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low-spin ferric heme iron (type II) in contrast to 17EE, which yields a high-spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to that of 17EE, with a different regioselectivity. Surprisingly, continuous-wave electron paramagnetic resonance (EPR) and HYSCORE EPR spectroscopy indicate that 17-click does not displace water from the sixth axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model in which 17-click pendant 1,2,3-TRZ hydrogen bonds with the sixth axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low-spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4 center dot 17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra.
  • Thumbnail Image
    Item
    The tetrahydrobiopterin radical interacting with high- and low-spin heme in neuronal nitric oxide synthase - A new indicator of the extent of NOS coupling
    (Elsevier, 2016) Krzyaniak, Matthew D.; Cruce, Alex A.; Vennam, Preethi; Lockart, Molly; Berka, Vladimir; Tsai, Ah-Lim; Bowman, Michael K.; University of Alabama Tuscaloosa; University of Texas Health Science Center Houston; Northwestern University; Linkoping University
    Reaction intermediates trapped during the single-turnover reaction of the neuronal ferrous nitric oxide synthase oxygenase domain (Fe(II)nNOS(OX)) show four EPR spectra of free radicals. Fully-coupled nNOSOX with cofactor (tetrahydrobiopterin, BH4) and substrate (L-arginine) forms the typical BH4 cation radical with an EPR spectrum similar to 4.0 mT wide and hyperfine tensors similar to reports for a biopterin cation radical in inducible NOSOX (iNOS(OX)). With excess thiol, nNOSox lacking BH4 and L-arg is known to produce superoxide. In contrast, we find that nNOSOX with BH4 but no L-arg forms two radicals with rather different, fast (similar to 250 mu s at 5 K) and slower (similar to 500 mu s at 20 K), electron spin relaxation rates and a combined similar to 7.0 mT wide EPR spectrum. Rapid freeze-quench CW- and pulsed-EPR measurements are used to identify these radicals and their origin. These two species are the same radical with identical nuclear hyperfine couplings, but with spin-spin couplings to high-spin (4.0 mT component) or low-spin (7.0 mT component) Fe(III) heme. Uncoupled reactions of nNOS leave the enzyme in states that can be chemically reduced to sustain unregulated production of NO and reactive oxygen species in ischemia-reperfusion injury. The broad EPR signal is a convenient indicator of uncoupled nNOS reactions producing low-spin Fe(III) heme.