Binding of quinidine radically increases the stability and decreases the flexibility of the cytochrome P450 2D6 active site

Pressure effects reveal that changes in the redox states of the heme iron complexes in the sensor domains of two heme‐based oxygen sensor proteins, EcDOS and YddV, have profound effects on their flexibility

“…the heme active site). The values of −α for the studied Ec DOS heme proteins were lower than those calculated for various Fe(II)–CO complexes of cytochromes P450, which ranged from 0.233 cm −1 ·MPa −1 for cytochrome P450 2A6 to 0.449 cm −1 ·MPa −1 for the more flexible cytochrome P450 3A4 .…”

“…and Table ). These results indicate that, like the EcDOS‐heme domain, the Fe(II)–CO form of the YddV‐heme is comparatively rigid and has appreciably lower −α values than determined for cytochrome P450 enzymes, which ranged from 0.233 to 0.449 as discussed above .…”

“…It is also essential to be aware of any spin equilibria that may occur, and the effects of the various substrates that may be present in the heme active site before performing such analyses. Therefore, previous studies on the active‐site flexibility of various cytochromes P450 focused on applying hydrostatic pressure to their Fe(II)–CO forms to ensure that the complexes were as well‐defined as possible, with six‐coordinate low‐spin heme iron centers . Under such conditions, the dependence of the position of the Soret band on the applied pressure may be described using a simple equation:where S is the position of the Soret band under high pressures (cm −1 ), P is the difference between the applied pressure and the pressure under ambient conditions (MPa), S 0 is the position of the Soret band under ambient conditions (cm −1 ), and the slope (α) is related to the compressibility of the heme active site .…”

Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery

“…Purification of P450 2D15 was carried out starting from the isolated membrane fraction using an anion exchange DEAE sepharose column (GE-healthcare, Italy) and followed by a nickel ion affinity chromatography step (GE-healthcare, Italy) where the protein remained bound through the engineered His-tag and subsequently eluted using a 0-40 mM linear gradient of histidine. In order to preserve the P450 2D15 stability the protein was purified in presence of the human P450 2D6 inhibitor quinidine [36]. The UV-visible spectra of the oxidized, reduced and reduced-carbon monoxide bound forms of the protein were recorded on a Hewlett-Packard 8453 diode array spectrophotometer.…”

“…Cytochrome P450 2D15 (57 kDa) (Fig.1A) was purified in presence of quinidine, a known inhibitor of human P450 2D6. The purified protein was also stored in the presence of this inhibitor which has been shown to preserve the stability of the protein [36]. The inhibitor was removed prior to each experimental assay through buffer exchange.…”