*2.4. Molecular Docking*

We used a CYP51A1 crystal structure Protein Data Bank (PDB) ID: 3LD6 for molecular docking. The resulting models were selected based on the higher values of scoring function. The obtained docking poses are shown in Figure 4. Based on the docking results, luteolin binds very close to the heme coordinating iron (less than 3 Å) by the 3-OH-group of the phenyl ring. In contrast, luteolin 7,3- -disulfate binds at >8.5 Å from the heme. The docking results are consistent with the spectral titration data—luteolin induces reverse type I spectra, while luteolin 7,3- -disulfate does not change the spectral response.

**Figure 4.** Luteolin and luteolin 7,3- -disulfate docked to the active site of human CYP51A1. The secondary structure of the protein is depicted as a ribbon and colored green. The amino acid side chains are shown as sticks and are colored in grey. The flavonoids and heme are shown as sticks and are colored in magenta and orange, respectively.

Asp231 (C-terminal part of the F-helix) H-bonded to luteolin and is important for the enzymatic activity of CYP51 [37]. The negative charge in this position is highly conserved in Prokaryotes and Eukaryotes [38]. Residues Leu310 (part of I-helix), Met378 and Ile379 (both K-helix–β1-4 loop) are involved in the interaction with luteolin 7,3- -disulfate. Residues Leu310 and Met378 are conservative among *Chordata*, and Ile379 is conservative among primates [38]. Notably, these structural elements were shown to interact with the elongated azole inhibitors (PDB ID: 3LD6, 4UHI and 6Q2T), suggesting that several residues of the active site are utilized for the distant binding of luteolin 7,3- -disulfate.

The docking pose obtained for luteolin 7,3- -disulfate showed binding in the access channel (Figure 4). Thus, the inhibition effect could be the result of blocking of the substrate access channel. However, the inhibition of CYP51A1 by luteolin 7,3- -disulfate does not exclude the modulation of interaction with its redox partner. The proximal surface of CYP51A1—where the redox partner, cytochrome P450 reductase, is binding—contains positively charged amino acids which can interact with the negatively charged sulfate groups of luteolin 7,3- -disulfate.
