Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.


Supporting Information Table of Contents
Page Table A Table B | Percentage occupancy of hydrogen bonds between the interface of P450c17 and cyt b5 during the molecular dynamics simulations S10 Figure H | QCM data S11 Choosing an electrode platform for probing the P450c17 and cyt b5 electrochemistry S12 Figure I | Electrocatalytic properties of P450c17 adsorbed on CNT S13 Figure J | Oxygen electroreduction catalyzed by protein-and hemin-modified CNT electrodes S14 Enzymatic capacity of P450c17 immobilized on a CNT-based electrode S15 Adsorption of cyt b5 on bare CNT-based electrodes S16 Figure K | Electrochemistry of cyt b5-polymyxin B films S17     Arg449 is not directly involved in a polar interaction, its side-chain is free to move, and in a fluid protein environment this residue would be expected to play a role in stabilizing the interface. S10 *Occupancy can be greater than 100% as a given residue pair may contain more than one hydrogen bond, each of which was counted separately.

Enzymatic capacity of P450c17 immobilized on a CNT-based electrode
Controlled potential electrolysis of the air-saturated aqueous 3 mM pregnenolone solution (0.20 M NaCl + 0.02 M (K 2 HPO 4 + KH 2 PO 4 )) was undertaken with the use of a composite electrode fabricated via adsorption of P450c17 on CNTs immobilized on a carbon cloth to test the enzymatic capacity of these P450c17-modified CNT-electrodes. Reductive currents monitored at the constant potential of -0.31 V, where O 2 electroreduction was predominantly catalyzed by the iron heme rather than by a carbon support, degraded to the values obtained with a protein-free electrode within 10 min under the employed electrolysis conditions (Fig. I).
Complete degradation of the iron heme catalytic species was confirmed by chronoamperometric and voltammetric measurements in air-saturated and deoxygenated solutions. Gas chromatograms (flame ionization detector) of the trimethylsilylated dichloromethane or methyl tert-butyl ether extracts from the electrolyzed pregnenolone solutions exhibited a peak at the retention time of trimethylsilylated 17α-hydroxy-pregnenolone as well as other peaks of unknown origin at slightly higher retention times, none of which were found in chromatograms obtained with the initial non-electrolyzed pregnenolone solution (Fig. I, panel c). The concentration of 17α-hydroxy-pregnenolone was below 10 -9 M, while the charge passed during the electrolysis provided two-electron reduction of 1.8·10 -5 M O 2 predominantly to H 2 O 2 . Most probably, hydrogen peroxide can assist enzymatic hydroxylation of pregnenolone by P450c17 as well as can chemically oxidize the steroid hormone substrate S(4-6, 8). Obviously, the latter pathway lacks regioselectivity and can result in the formation of the unidentified compounds, which were detected chromatographically in the electrolyzed pregnenolone solution as peaks in the vicinity of the trimethylsilylated 17α-hydroxy-pregnenolone signal. The ability to detect 17αhydroxy-pregnenolone in solutions electrolyzed using P450c17/CNT electrodes provide confidence that immobilization of P450c17 is functionally competent and catalytically active during the electrochemical experiments.

Adsorption of cyt b5 on bare CNT-based electrodes
Although the cyt b5 contains an abundance of negatively charged residues (as confirmed by voltammetric studies on cyt b5-polymyxin B complexes immobilized on PGE electrodes shown in Fig. K), adsorption of both wild type and E48G/E49G mutant cyt b5 proteins on CNT electrodes produced pronounced voltammetric signals from the surface confined iron heme ( Fig. 2 of the main text). Typically, the surface concentration of the Fe-containing species on the CNT electrodes obtained with E48G/E49G cyt b5 was higher than observed after adsorption of wt cyt b5, as might be expected when the surface-exposed glutamic acid residues were replaced with glycine. Moreover, the Γ values attained upon modification of a CNT electrode with cyt b5 were ca 2-3 times higher than those measured with P450c17 electrode.
The mechanism of adsorption of cyt b5 on CNT electrodes is unknown. The apparent reversible potentials for the Fe 3+/2+ process derived from the a.c. voltammetric analysis of CNT electrodes modified with wt and E48G/E49G cyt b5 differ from each other and, most importantly, from !" ! for pure hemin ( Table 1 in the main text). Furthermore, these differences in !" ! follow the same tendency when wild type and mutant proteins were adsorbed on pure CNTs or connected to a PGE electrode via polymyxin B, i.e. under conditions where denaturation was less likely (cf. data in Table 1 in the main text and Table C). Substantial difference in !" ! obtained with cyt b5modified CNT and polymyxin B/PGE electrodes can be explained by the formation of complexes between polymyxin B and proteins, presumably, via the carboxylic functional groups of the iron heme S(9). Alternatively, CNT electrodes modified with cyt b5 exhibit pronounced catalytic activity for oxygen reduction (Fig. J), notwithstanding the coordination of the heme iron atom to two histidine residues, that would preclude access of molecular oxygen S(10). A similar effect was reported by Zhang et al. S(11) when cytochrome c 3 was adsorbed on a glassy carbon electrode. Thus, the cyt b5 proteins could be partially disrupted upon adsorption on CNTs in order to provide access of molecular oxygen to the iron atom of the heme moiety. However, there are no circumstances we can envisage that affects the major conclusions of the study on interactions between P450c17 and wild type or E48G/E49G cyt b5 proteins.