Tight binding of cytochrome b5 to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis

Abstract

It has been recognized for >50 years that cytochrome b 5 ( b 5 ) stimulates some cytochrome P450 (P450)–catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b 5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b 5 function. One of the issues in studying b 5 –P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b 5 that can be used in titrations. The labeled b 5 bound to WT P450 17A1 with a K d of 2.5 nM and rapid kinetics, on the order of 1 s −1 . Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b 5 binding. K d values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C- b 5 fluorescence at higher concentrations. The addition of NADPH–P450 reductase (POR) to an Alexa 488-T70C- b 5 :P450 17A1 complex resulted in a concentration-dependent partial restoration of b 5 fluorescence, indicative of a ternary P450: b 5 :POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1: b 5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

Cytochrome b5 Binds Tightly to Several Human Cytochrome P450 Enzymes

“…Rationale. In recent work with P450 17A1 (Kim et al, 2021) we labeled a human b 5 mutant (T70C) with a dye, based on a previous approach used to monitor the binding of bacterial P450 cam and rat b 5 (Stayton et al, 1988;Stayton et al, 1989). The site of attachment is removed from the putative sites of binding (Glu-48, Glu-49), at least to P450 17A1 (Naffin-Olivos and Auchus, 2006), on the opposite side of the heme prosthetic group (Fig.…”

“…All have been found to be catalytically active under appropriate reconstitution conditions and, in several cases, to show b 5 stimulation (Yamazaki et al 2002). Alexa 488-T70C-b 5 was prepared as described elsewhere in a study of P450 17A1 (Kim et al, 2021). All proteins were of high purity as judged by SDS-gel electrophoresis and did not contain any obvious cleavage products.…”

“…1) (Stayton et al, 1988), conjugated it with a fluorescent dye (Alexa 488 maleimide) (Fig. 2), and utilized this probe in studies on the interaction of b 5 with P450 17A1 (Kim et al, 2021). The results were interpreted in a model of very tight binding of b 5 and P450 17A1, with POR binding to form a ternary complex during catalysis (Kim et al, 2021).…”

“…2), and utilized this probe in studies on the interaction of b 5 with P450 17A1 (Kim et al, 2021). The results were interpreted in a model of very tight binding of b 5 and P450 17A1, with POR binding to form a ternary complex during catalysis (Kim et al, 2021). We have now extended this approach to other human P450s, particularly those with precedents for stimulation by b 5 .…”

Optimization of Multiparameters for Increased Yields of Cytochrome B5 in Bioreactors

“…The production of cyt b5 in bioreactors may provide sufficient amounts for its application in several research and development studies, namely: In vitro studies of lipid-protein interactions [ 18 ]; determination of the effect of allosteric alteration of cyt b5 active site conformation [ 19 ]; studies regarding cyt b5 interactions with cytochrome P450 enzymes aimed at activation of pharmaceutical drugs [ 20 , 21 ]; drug metabolism and biosynthesis of natural products by the P450-cyt b5 complex and development of corresponding pharmacokinetic models to predict in vivo action in the early stages of drug discovery [ 22 , 23 ]. Cyt b5 has also been successfully used as an efficient affinity matrix for the purification of electron-transfer proteins [ 24 ], and as a model protein for developing batch and continuous extraction processes [ 25 ].…”