Abstract
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.
Highlights
Cytochrome P450 (P450) enzymes are ubiquitous throughout nature, utilizing a heme moiety at their active site with the electrons delivered via a protein redox pathway for hydroxylation of substrates [1]
In order to be able to identify faradaic response arising from loss of the heme moiety from either the P450c17 or the cyt b5 proteins in our experiments, we initially studied the voltammetry of free hemin adsorbed on carbon nanotubes (CNT)-based electrodes
The fluorescence resonance energy transfer (FRET) analyses in living cells showed a heterodimeric interaction between P450c17 and cyt b5 and were conducted [22], but not reported previously, as part of an examination of steroidogenic P450 enzyme interactions
Summary
Cytochrome P450 (P450) enzymes are ubiquitous throughout nature, utilizing a heme moiety at their active site with the electrons delivered via a protein redox pathway for hydroxylation of substrates [1]. Our current understanding of mammalian P450 biochemistry is derived mostly from studies on hepatic P450 enzymes. These differ from steroidogenic P450s in two fundamental ways. The hepatic P450s typically catalyze mono-hydroxylations needed to solubilize xenobiotics, required to assist with their excretion from the body. PLOS ONE | DOI:10.1371/journal.pone.0141252 November 20, 2015
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