CYP116B5hd, a self-sufficient P450 cytochrome: A dataset of its electronic and geometrical properties

Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study

“…In the CYP116B5hd active site, the Fe III is coordinated by the four nitrogen atoms of the heme ring, forming a very stable complex; in addition to that, a water molecule and a cysteine residue act as axial ligands. The octahedral geometry and the strong ligands coordinating the Fe III define a low-spin electron state ( S = ½), with the only unpaired electron of the ion dwelling in an orbital, which results from the mixing of the t 2g d orbitals caused by spin–orbit coupling [ 33 , 34 , 35 , 64 , 65 ]. Following this model, the g -values obtained from the CW-EPR spectra of the enzyme and of low-spin hemeproteins, in general, can be related to the energy of the t 2g orbitals of the iron considering a one-electron model (or rather a “hole”).…”

“…Both imidazole-coordinated species have, within the error limits, the same ∆/ξ parameter. The V/ξ values, accounting for the lack of axiality in the spin system since it measures the energy difference between the d zx and d zy orbitals, are different for the two species, which, as we demonstrate here, is due to the imidazole plane adopting a different orientation with respect to the porphyrin ring [ 32 , 33 ].…”

“…Labeling the nuclei of the axial distal ligand, H 2 O or imidazole, with isotopes 2 H and 15 N, did not have any effect on the CW-EPR spectra, and consequently, on the crystal field parameters ∆ and V. Therefore, one can conclude that the hyperfine couplings of the iron with these nuclei are smaller than other line-broadening mechanisms. To access the information about the hyperfine interactions occurring between the iron spin and the magnetic nuclei present in its environment, we previously tested the suitability of HYSCORE experiments, which showed couplings with several nuclei in the active site of the protein [32,33]. To fully characterize the anisotropy of the hyperfine and nuclear quadrupole interactions, HYSCORE experiments were recorded at different values of the magnetic field spanning the whole EPR spectrum of the protein, both with and without imidazole.…”

“…Since CYP450s possess a characteristic active site with an Fe III -heme center as the fulcrum, and given the paramagnetic nature of the iron, not only in the resting state of the enzyme but also in several intermediates generated during the CYP450 catalytical cycle, EPR spectroscopy lends itself as a very suitable technique to characterize and analyze this system [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. In our previous work [ 32 , 33 ], we carried out a study on CYP116B5hd, where we analyzed the g -values according to the electronic model for low-spin Fe III by Taylor [ 34 , 35 ] and compared our results with those obtained for other classical (monooxygenase-like) CYP450s, such as CYP102A1 (CYPBM3), and more exotic (peroxygenase-like) CYP450s, such as CYP152B1, CYP152K6, and CYP152L1. From this comparison, it was concluded that the electronic state of the enzyme was very much like the classic P450, determined by the active site in the close proximity of iron.…”

Catalytically self‐sufficient CYP116B5: Domain switch for improved peroxygenase activity

“…[29] Subsequently, its heme domain (CYP116B5-hd) was expressed, purified, and characterized, revealing a high tolerance to H 2 O 2 and an extraordinary ability to work as a peroxygenase by using the peroxide shunt, [30,31] despite maintaining the characteristics of a monooxygenase. [32,33] The recombinant production of the heme domain represented an advantage also in terms of solubility, stability, and production yield. On the other hand, although the ability to carry out the catalysis via the peroxide shunt using H 2 O 2 instead of NAD(P)H represents a great advantage in terms of costs, the comparison of the catalytic parameters with its full-length form was still missing.…”