MOTIVATION: Cytochrome P450 proteins play a crucial role in human metabolism, ranging from hormone production to drug metabolism. While multiple commonly known variants have known effects on the individual cytochrome P450 protein performance, the pathogenicity information is usually experimentally limited to only a few mutations. Current pathogenicity prediction software enables the extension of the scope to virtually mutate all amino acids with all possible substitutional mutations. In this work, we do a comprehensive exploration that unveils pathogenicity patterns in the human cytochrome P450 family. Pathogenicity analysis was conducted across proteins using SIFT, AlphaMissense, and PrimateAI-3D algorithms. RESULTS: Our findings indicate a progressive increase in pathogenicity along protein tunnels-identified via MOLE-toward the cofactor binding site, underscoring the essential role of cofactor interactions in enzymatic function. Notably, the integrity of tunnels and cofactor environment emerges as a critical factor, with even single amino acid alterations potentially disrupting molecular guidance to active sites. These insights highlight the fundamental role of structural pathways in preserving cytochrome P450 functionality, with implications for understanding disease-associated variants and drug metabolism. AVAILABILITY AND IMPLEMENTATION: Data and source code can be found at https://github.com/annaspac/P450_pathogenicity_codes.

CEITEC Central European Institute of Technology Masaryk University Brno Brno 625 00 Czech Republic

Department of Physical Chemistry Faculty of Science Palacký University Olomouc 771 46 Czech Republic

IT4Innovations VSB Technical University of Ostrava Ostrava Poruba 708 00 Czech Republic

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