Structural and Spectroscopic Characterization of Self-Sufficient Cytochrome P450 Fusion Enzymes: CYP102A1 and CYP116B
Abstract
Cytochrome P450 (CYP) monooxygenases are heme-thiolate enzymes that catalyze diverse oxidative transformations essential to metabolism, detoxification, and biosynthesis. While most CYPs rely on separate redox partner proteins for electron delivery, a subset-known as self-sufficient or fusion P450s-incorporate the reductase domain(s) directly within a single polypeptide, enabling efficient intramolecular electron transfer and, in some cases, exceptionally high catalytic turnover. This review examines two representative self-sufficient P450 systems, CYP102A1 (P450 BM3) and CYP116B, summarizing spectroscopic evidence (UV-Vis, EPR, and resonance Raman), supported by available crystal structures, to compare how their distinct domain architectures shape heme electronic structure and catalytic mechanism. CYP102A1, a fused FAD/FMN-heme flavocytochrome from Bacillus megaterium, undergoes pronounced substrate-induced conformational rearrangement and low-spin/highspin switching, properties that are further tunable through active-site mutagenesis (e.g., A82F, F87V, F393 variants) and that underlie its status as the most extensively studied and engineered P450 scaffold. In contrast, CYP116B enzymes, which transfer electrons through a defined FMN-[2Fe–2S]-heme pathway, maintain a comparatively rigid, conserved low-spin heme environment that is less dynamically coupled to substrate binding, although engineered variants (e.g., CYP116B5-SOX) demonstrate their potential for peroxide-driven biocatalysis. Together, these comparisons illustrate how differences in fusion architecture translate into distinct strategies for electron transfer and catalytic control, offering complementary design principlesstructural plasticity in BM3 versus a stable redox scaffold in CYP116B-for engineering nextgeneration self-sufficient P450 biocatalysts.
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BibTeX
@misc{vungarala2026structural,
title = {{Structural and Spectroscopic Characterization of Self-Sufficient Cytochrome P450 Fusion Enzymes: CYP102A1 and CYP116B}},
author = {Sravya Vungarala and Mason Daras and Yasmine Elshoweikh and Yilin Liu},
howpublished = {ChemRxiv},
year = {2026},
month = sep,
doi = {10.26434/chemrxiv.15009513/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009513/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Structural and Spectroscopic Characterization of Self-Sufficient Cytochrome P450 Fusion Enzymes: CYP102A1 and CYP116B
AU - Sravya Vungarala
AU - Mason Daras
AU - Yasmine Elshoweikh
AU - Yilin Liu
PY - 2026
DA - 2026/09/28
PB - ChemRxiv
DO - 10.26434/chemrxiv.15009513/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009513/v1
AB - Cytochrome P450 (CYP) monooxygenases are heme-thiolate enzymes that catalyze diverse oxidative transformations essential to metabolism, detoxification, and biosynthesis. While most CYPs rely on separate redox partner proteins for electron delivery, a subset-known as self-sufficient or fusion P450s-incorporate the reductase domain(s) directly within a single polypeptide, enabling efficient intramolecular electron transfer and, in some cases, exceptionally high catalytic turnover. This review examines two representative self-sufficient P450 systems, CYP102A1 (P450 BM3) and CYP116B, summarizing spectroscopic evidence (UV-Vis, EPR, and resonance Raman), supported by available crystal structures, to compare how their distinct domain architectures shape heme electronic structure and catalytic mechanism. CYP102A1, a fused FAD/FMN-heme flavocytochrome from Bacillus megaterium, undergoes pronounced substrate-induced conformational rearrangement and low-spin/highspin switching, properties that are further tunable through active-site mutagenesis (e.g., A82F, F87V, F393 variants) and that underlie its status as the most extensively studied and engineered P450 scaffold. In contrast, CYP116B enzymes, which transfer electrons through a defined FMN-[2Fe–2S]-heme pathway, maintain a comparatively rigid, conserved low-spin heme environment that is less dynamically coupled to substrate binding, although engineered variants (e.g., CYP116B5-SOX) demonstrate their potential for peroxide-driven biocatalysis. Together, these comparisons illustrate how differences in fusion architecture translate into distinct strategies for electron transfer and catalytic control, offering complementary design principlesstructural plasticity in BM3 versus a stable redox scaffold in CYP116B-for engineering nextgeneration self-sufficient P450 biocatalysts.
ER -