Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study
The cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synth...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-01-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/29/2/518 |
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| author | Antonino Famulari Danilo Correddu Giovanna Di Nardo Gianfranco Gilardi George Mitrikas Mario Chiesa Inés García-Rubio |
| author_facet | Antonino Famulari Danilo Correddu Giovanna Di Nardo Gianfranco Gilardi George Mitrikas Mario Chiesa Inés García-Rubio |
| author_sort | Antonino Famulari |
| collection | DOAJ |
| description | The cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synthesis using the peroxide shunt. In this meticulous study, we use hyperfine spectroscopy with a multifrequency approach (X- and Q-band) to characterize in detail the electronic structure of the heme iron of CYP116B5hd in the resting state, which provides structural details about its active site. The hyperfine dipole–dipole interaction between the electron and proton nuclear spins allows for the locating of two different protons from the coordinated water and a beta proton from the cysteine axial ligand of heme iron with respect to the magnetic axes centered on the iron. Additionally, since new anti-cancer therapies target the inhibition of P450s, here we use the CYP116B5hd system—imidazole as a model for studying cytochrome P450 inhibition by an azo compound. The effects of the inhibition of protein by imidazole in the active-site geometry and electron spin distribution are presented. The binding of imidazole to CYP116B5hd results in an imidazole–nitrogen axial coordination and a low-spin heme Fe<sup>III</sup>. HYSCORE experiments were used to detect the hyperfine interactions. The combined interpretation of the gyromagnetic tensor and the hyperfine and quadrupole tensors of magnetic nuclei coupled to the iron electron spin allowed us to obtain a precise picture of the active-site geometry, including the orientation of the semi-occupied orbitals and magnetic axes, which coincide with the porphyrin N-Fe-N axes. The electronic structure of the iron does not seem to be affected by imidazole binding. Two different possible coordination geometries of the axial imidazole were observed. The angles between <i>g<sub>x</sub></i> (coinciding with one of the N-Fe-N axes) and the projection of the imidazole plane on the heme were determined to be −60° and −25° for each of the two possibilities via measurement of the hyperfine structure of the axially coordinated <sup>14</sup>N. |
| first_indexed | 2024-03-08T09:49:01Z |
| format | Article |
| id | doaj.art-aec690c1c7d74af6be52c350ade4db69 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-08T09:49:01Z |
| publishDate | 2024-01-01 |
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| spelling | doaj.art-aec690c1c7d74af6be52c350ade4db692024-01-29T14:09:19ZengMDPI AGMolecules1420-30492024-01-0129251810.3390/molecules29020518Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) StudyAntonino Famulari0Danilo Correddu1Giovanna Di Nardo2Gianfranco Gilardi3George Mitrikas4Mario Chiesa5Inés García-Rubio6Departamento de Física de la Materia Condensada, Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza, SpainDepartment of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Torino, ItalyDepartment of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Torino, ItalyDepartment of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Torino, ItalyInstitute of Nanoscience and Nanotechnology, NCSR Demokritos, 15341 Athens, GreeceDepartment of Chemistry, University of Turin, Via Giuria 9, 10125 Torino, ItalyDepartamento de Física de la Materia Condensada, Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza, SpainThe cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synthesis using the peroxide shunt. In this meticulous study, we use hyperfine spectroscopy with a multifrequency approach (X- and Q-band) to characterize in detail the electronic structure of the heme iron of CYP116B5hd in the resting state, which provides structural details about its active site. The hyperfine dipole–dipole interaction between the electron and proton nuclear spins allows for the locating of two different protons from the coordinated water and a beta proton from the cysteine axial ligand of heme iron with respect to the magnetic axes centered on the iron. Additionally, since new anti-cancer therapies target the inhibition of P450s, here we use the CYP116B5hd system—imidazole as a model for studying cytochrome P450 inhibition by an azo compound. The effects of the inhibition of protein by imidazole in the active-site geometry and electron spin distribution are presented. The binding of imidazole to CYP116B5hd results in an imidazole–nitrogen axial coordination and a low-spin heme Fe<sup>III</sup>. HYSCORE experiments were used to detect the hyperfine interactions. The combined interpretation of the gyromagnetic tensor and the hyperfine and quadrupole tensors of magnetic nuclei coupled to the iron electron spin allowed us to obtain a precise picture of the active-site geometry, including the orientation of the semi-occupied orbitals and magnetic axes, which coincide with the porphyrin N-Fe-N axes. The electronic structure of the iron does not seem to be affected by imidazole binding. Two different possible coordination geometries of the axial imidazole were observed. The angles between <i>g<sub>x</sub></i> (coinciding with one of the N-Fe-N axes) and the projection of the imidazole plane on the heme were determined to be −60° and −25° for each of the two possibilities via measurement of the hyperfine structure of the axially coordinated <sup>14</sup>N.https://www.mdpi.com/1420-3049/29/2/518EPR spectroscopyCYP450HYSCOREperoxygenasehyperfine interactionslow-spin hemeprotein |
| spellingShingle | Antonino Famulari Danilo Correddu Giovanna Di Nardo Gianfranco Gilardi George Mitrikas Mario Chiesa Inés García-Rubio Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study Molecules EPR spectroscopy CYP450 HYSCORE peroxygenase hyperfine interactions low-spin hemeprotein |
| title | Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study |
| title_full | Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study |
| title_fullStr | Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study |
| title_full_unstemmed | Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study |
| title_short | Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study |
| title_sort | heme spin distribution in the substrate free and inhibited novel cyp116b5hd a multifrequency hyperfine sublevel correlation hyscore study |
| topic | EPR spectroscopy CYP450 HYSCORE peroxygenase hyperfine interactions low-spin hemeprotein |
| url | https://www.mdpi.com/1420-3049/29/2/518 |
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