Abstract
Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called “uncoupling”, whereby NAD(P)H derived electrons are lost due to the reduced intermediates’ (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2–22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.
Highlights
Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs)
C. tropicalis possesses two genes coding for CPR, cpr-a and cpr-b24
Analysis of the C. tropicalis CPR (CtCPR) sequence revealed the presence of a single membrane-spanning region comprising the first 22 amino acids of the protein and in an attempt to produce more soluble CPR, a truncated, untagged version (Δ2–22) of the enzyme was expressed in E. coli
Summary
Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs) It can reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. The proposed mechanism is that during inter-flavin electron transfer, the enzyme is in a compact, closed conformation which facilitates the electron flow between the prosthetic groups This conformation is not ideal for interaction with protein acceptors and CPR undergoes conformational changes to expose the reduced FMN for interaction with CYPs5. The overall mechanism and details of the CPR interaction with its redox acceptors remain elusive and further analysis is necessary to completely understand the dynamics of electron shuttle to the CYPs. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called uncoupling phenomenon, which generates reactive oxygen species (ROS). ROS result in loss of electrons and leads to an inefficient reaction, oxidative damage and inactivates the enzymes[19]
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