*2.1. Surface Plasmon Resonance*

The CYP51A1 complex formation with flavonoids was detected using a SPR-biosensor. Lanosterol was used as a positive control to confirm the ability of immobilized CYP51A1 to bind ligands. With the CYP51A1 immobilized on the biosensor chip surface, we were able to detect the interaction with baicalein, luteolin and luteolin 7,3- -disulfate (Figure 2).

**Figure 2.** Typical surface plasmon resonance sensorgrams of binding between immobilized CYP51A1 on the optical chip and baicalein, luteolin and luteolin 7,3- -disulfate at different concentrations: 10 (1), 25 (2), 50 (3), 75 (4) and 100 μM (5). Fitting curves (theoretical models) are highlighted in black; Chi2 = 25.3 (baicalein), 68.2 (luteolin), 10.2 (luteolin 7,3- -disulfate).

The equilibrium dissociation constant (Kd) values of CYP51A1/flavonoid complexes were in the range of 2.9–20 μM, calculated association and dissociation rate constants are shown in Table 1. The obtained Kd value of the CYP51A1/lanosterol complex was 2.4 μM, which is comparable with the previously published data [35]. The association rate of the CYP51A1 complex with luteolin 7,3- -disulfate is seven times faster compared

to the complex formation with lanosterol, while the dissociation rate is about eight times higher. The resulting Kd value for both complexes is similar. CYP51A1 complex formation with baicalein and luteolin is characterized by the increased association rate compared to lanosterol, but the main differences in the resulting Kd value are due to the great increase in dissociation rates of the complexes. Overall, the binding of flavonoids is faster compared to the natural substrate, but the dissociation of the complexes is faster as well. The highest affinity was detected for luteolin 7,3- -disulfate, which is more soluble.

**Table 1.** Kinetic and equilibrium parameters of cytochrome P450(51) (CYP51A1) complex formation with lanosterol, baicalein, luteolin and luteolin 7,3- -disulfate.


The table shows the average values of the parameters ± standard deviation, n = 3.

#### *2.2. Spectral Titration Analysis*

The difference spectra of CYP51A1 were obtained by titration with baicalein, luteolin and luteolin 7,3- -disulfate in the presence of lanosterol. Baicalein and luteolin induced a reverse type I spectral response with absorbance minimum at 390 nm and maximum at 420 nm for luteolin and 436 nm for baicalein (Figure 3). These spectral changes are consistent with the previously detected interaction of cytochrome P450 1B1 with compounds of flavonoid class [36]. Titration with luteolin 7,3- -disulfate (up to 30 μM) does not cause changes in the difference spectrum of CYP51A1. The apparent dissociation constant (Kdapp) values of the complexes of CYP51A1 with baicalein and luteolin were 8.2 ± 0.4 and 5.1 ± 0.5 μM, respectively. It should be noted that the Kd values from spectrophotometric titration experiments differ from those obtained using SPR. These differences can be attributed to the different affinities of the complexes in solution and immobilized on the surface of the optical chip. Interaction with the different sites of the enzyme cannot be excluded during SPR measurements and the measured Kd reflects all possible interactions between the ligand and enzyme, while spectral assays detect interactions of ligand only within close vicinity of the heme cofactor buried in the CYP active site.

**Figure 3.** Difference spectra of CYP51A1 in the presence of lanosterol after addition of baicalein (up to 30 μM) and luteolin (up to 15 μM). The arrows indicate the direction of the spectral changes with increasing ligand concentration.
