Direct electrochemistry of human, bovine and porcine cytochrome P450c17

Abstract

The direct electrochemistry of human, bovine and porcine cytochrome P450c17 (CYP17) has been examined on an edge-oriented pyrolytic graphite electrode. The recombinant protein was immobilized on an electrode modified with a surfactant to simulate the environment of a biological membrane, and hence physiological electron-transfer conditions. The P450 enzymes all retained 'electron-transfer' activity while immobilized at the electrode surface as assessed by the presence of catalytic signals under aerobic conditions. The redox potentials for porcine P450c17 were more positive (anodic) than both the human and bovine forms, perhaps reflecting the differences in substrate specificity for these species. In addition, these enzymes were all influenced by pH, consistent with a single proton associated with the single electron-transfer event. Ionic strength of the buffer medium also shifted the redox potentials towards positive, suggesting that electrostatic forces contribute to the protein environment required for the electron-transfer process. The effect of substrate on the redox potential for each P450c17 was measured in the presence of pregnenolone, progesterone, 17 -hydroxypregnenolone and 17 -hydroxyprogesterone. However, no influence on the redox parameters was observed.

Control by Substrate of the Cytochrome P450-Dependent Redox Machinery: Mechanistic Insights

“…Thus, liganding to the recombinant human enzyme of pregnenolone and progesterone together with their respective 17 -hydroxy derivatives displayed K d values ranging from 1.4 to 5.3 μM accompanied by a considerable yield in high-spin conversion of the hemoprotein [175]. Even so, direct electrochemistry of the P450 from human, bovine and porcine sources immobilized on an edge-oriented pyrolytic graphite electrode failed to demonstrate any effect of these substrates/metabolic intermediates on the midpoint potential of CYP17 [176]. Moreover, turnover numbers were moderate [175], with the ratio of 17 -hydroxylase to C 17 -C 20 desmolase activity being modulated by the presence of b 5 [177].…”

“…Also, the V max /K m value for deoxycorticosterone conversion to aldosterone is about 47% that found for transformation of the steroid to corticosterone [174], negating the possibility of the rate-limiting steps in the two metabolic branches to be identical. Furthermore, steroid-induced changes in the redox properties of CYP17 are obviously not required to support hydroxylase/lyase activity in hormone synthesis [176].…”

“…[44,45,66], and a number of counter examples also come from exploration of the redox behaviour of other P450s from microbial phyla (vide supra). Moreover, careful evaluation of the conflicting data for vertebrate-type proteins [15,25,112,122,176] suggests extreme caution as to generalizations regarding the importance of spin/redox control in substrate turnover. This view dovetails nicely with results obtained with more physiological experimental settings: Introduction of substrate during liver perfusion under aerobic conditions rather invokes a slight cathodic shift of E 0' [102].…”

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

“…Canis familiaris is one of the most widely studied animal models used in safety determination of new pharmaceuticals [11][12] and, although the major isoforms of the human cytochromes P450 2D6, 3A4, 2E1, 2C19 and 1A2 have been identified in C. familiaris [13][14][15][16], there is still a lack of knowledge on their pharmacogenomic/metabolic diversity [9]. Electrochemical techniques already developed in our lab for human hepatic monooxygenases including cytochromes P450 [17][18][19][20][21][22] represent the ideal approach for a sensitive, accurate and rapid evaluation of animal P450-drug interactions obviating both the requirement for a redox partner and the addition of NADPH cofactor as already reported for some animal P450 enzymes recombinantly expressed in a soluble form [23][24][25]. In this work, Canis familiaris P450 2D15 was chosen as a model for the investigation of canine cytochromes P450 by adopting electrochemical approaches to provide a method for the screening of the safety of new chemical entities/drugs.…”

Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine, Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase

“…It is of great interest that P450c17 from different species appear to have different enzymatic activities (Buczko et al, 1995;Chung et al, 1987;Conley et al, 1992;Conley and Bird, 1997;Fevold et al, 1989;Lin et al, 1994;Namiki et al, 1988;Ono et al, 1988;Picodo-Leonard and Miller, 1987;Provencher et al, 1992;Simpson et al, 1987;Swart et al, 1993;Tremblay et al, 1994;Zuber et al, 1986). The factors that regulate these different activities of the P450c17 and the possible relationship of these differences to the protein structure that may differentiate enzymatic activities have been the topic of numerous investigations (Arlt et al, 2002;Brock and Waterman, 1999;Gilep et al, 2003;Gilep et al, 2004;Johnson et al, 2006;Lee-Robichaud et al, 1997;Lewis et al, 1998;Mathieu et al, 2000). In addition, cytochrome b 5 (b 5 ) has been identified as an important modulator of C17,20-lyase activity of porcine P450c17 enzyme (Katagiri et al, 1982;Onoda and Hall, 1982) and this has since been confirmed in reconstitution experiments in several laboratories with various P450c17 enzymes (Auchus et al, 1998;Brock and Waterman, 1999;Katagiri et al, 1995;Lee-Robichaudet et al, 1995Miller and Auchus, 2000).…”